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Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata Xiaoyu Li a,b , Hongxiang Sun c , Yiping Ye b , Fengyang Chen b , Jue Tu c , Yuanjiang Pan a,∗ a b c
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated
Received 15 September 2005
from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double
Received in revised form 29 March
bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic
2006
data and chemical evidence. These isolated compounds were assayed for their immunolog-
Accepted 17 April 2006
ical activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced
Published on line 21 June 2006
proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing
Keywords:
activities. © 2006 Elsevier Inc. All rights reserved.
Stephanotis mucronata Asclepiadaceae Immunomodulating activities
1.
Introduction
Immunosuppressive agents are the mainstay treatment for patients that have received organ grafts and are becoming increasingly important in the treatment of autoimmune diseases. There are, however, many problems with both the concept and reality of long-term immunosuppression as a therapeutic modality, both in terms of the nonspecific toxicity of the drugs that are currently available and the increased risk of infections and tumors arising from global suppression of the immune system. The immunosuppressant with less side effects is still a challenge to the medical system. C-21 steroidal glycosides have attracted much attention for their antitumor, antiepilepsy, and antifertility activities, and many such compounds have been isolated from plants, especially from those of the Asclepiadaceae family [1–4]. The dried roots of Stephanotis mucronata (Blanco) Merr. (Asclepi-
∗
adaceae) are used for the treatment of rheumatoid arthritis and rheumatic aches in Chinese folk medicine. We have earlier reported the isolation and structural elucidation of 12 pregnane glycosides: mucronatosides A–D, and stephanoside E from the stems of S. mucronata [5,6], and stemucronatosides A–G from the roots of S. mucronata [7,8]. We have also reported the immunomodulating activity of stemucronatosides A–G [7,8]. Among those compounds, stemucronatosides A–C, and F showed immunomodulating activities, while stemucronatosides D, E, and G showed immunosuppressive activities in a dose-dependent manner. In this paper, the chemical studies on the CHCl3 soluble extract from the stems of this plant were further undertaken, leading to four new immunomodulating C-21 steroidal glycosides: mucronatosides E–H (1–4) (Fig. 1). These results might be responsible, at least in part, for the treatment of rheumatoid arthritis and rheumatic aches in folk.
Corresponding author. Tel.: +86 571 879 51264; fax: +86 571 879 51264. E-mail address: [email protected] (Y. Pan). 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.04.007
684
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
2.2.
Plant material
The stems of S. mucronata were obtained from Yueqing, Zhejiang province, China. A voucher specimen (No. 200208) was identified by Prof. Zhang Zhi-Guo and deposited in the Laboratory of Natural Products Chemistry, Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, China.
2.3.
Extraction and isolation
The dried stems of S. mucronata (6.0 kg) were ground and extracted three times with 95% EtOH under reflux for 2 h. The extracts were evaporated in vacuo to give the EtOH extract (570 g). The ethanolic extract was partitioned with CHCl3 under reflux. Concentration of the CHCl3 extract yielded a yellow residue (223.5 g). The CHCl3 extract was partitioned with hexane under reflux. The hexane insoluble portion corresponded to a crude glycoside (193.5 g). The crude glycoside was subjected to CC (silica gel, gradient CHCl3 /MeOH 40:1 → 5:1, v/v) to give 8 main fractions. Fr. 3 (6.2 g) was subjected to CC (Rp-18, MeOH/H2 O 1:1 → 2:1, v/v; then HPLC, MeOH/H2 O 70:30, v/v) to give 4 (135 mg). Fr. 4 (9.4 g) was subjected to CC (Rp-18, MeOH/H2 O 1:1 → 2:1, v/v; then HPLC, MeOH/H2 O 67:33, v/v) to give 1 (155 mg), 2 (142 mg), and 3 (86 mg).
2.3.1.
Fig. 1 – The chemical structures of compounds 1–4.
2.
Experimental
2.1.
General methods
Melting points were determined on an MRK Prism Scope instrument and are uncorrected. Optical rotations were measured in MeOH with a Rudolph Research Autopol IV polarimeter. UV spectra were performed on a TU-1800PC spectrometer. IR spectra were recorded in a KBr pellet on a Perkin-Elmer 577 spectrometer. 1 H NMR, 13 C NMR, DEPT, 1 H, 1 H COSY, HMQC, and HMBC spectra were recorded at 500 MHz for 1 H, and at 125 MHz for 13 C, with a Bruker DRX 500 instrument in pyridined5 solution. HRESI-MS spectra were recorded on a Bruker Daltonics FTMS APEX III mass spectrometer. ESI-MS spectra were recorded on a Bruker Esquire 3000plus mass spectrometer. TLC was performed on precoated Kieselgel 60 F254 plates and Rp C18 (Merck), and the detection was achieved by spraying with 10% H2 SO4 followed by heating. Separation and purification were performed by column chromatography on silica gel (200–300 mesh, Qingdao), Rp C18 (Merck), and on semi-prep HPLC using an Agilent 1100 instrument (Zorbax column 9 mm × 250 mm, DAD).
Mucronatoside E (1)
C48 H78 O21 : white amorphous powder; mp 155–158◦ ; ◦ (MeOH; ca. 0.1); UV (MeOH) [˛]20 max : 207.0 nm; D + 34.1 IR (KBr) max : 3510 (OH), 1701 (C O), 1640 (C C), 1165 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1013.4923 ([C48 H78 O21 + Na]+ , calcd. 1013.4928). ESI-MS (positive) m/z: 1013.4 [M + Na]+ , 851.4 [M + Na − 162]+ , 691.3 [M + Na − 162-160]+ , 547.2 [M + Na − 162-160-144]+ , 403.1 [M + Na − 162 − 160-144-144]+ , 633.2 [M + Na − 380]+ , 489.2 [M + Na − 380-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.2.
Mucronatoside F (2)
C48 H80 O21 : white amorphous powder; mp 165–167◦ ; [˛]20 D + 18.0◦ (MeOH; ca. 0.1); UV (MeOH) max : 207.0 nm; IR (KBr) max : 3480 (OH), 1640 (C C), 1165 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1015.5103 ([C48 H80 O21 + Na]+ , calcd. 1015.5084). ESIMS (positive) m/z: 1015.4 [M + Na]+ , 853.4 [M + Na − 162]+ , 693.3 [M + Na − 162-160]+ , 549.3 [M + Na − 162-160-144]+ , 633.2 [M + Na − 382]+ , 489.1 [M + Na − 382-144]+ , 345.1 [M + Na − 382144-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.3.
Mucronatoside G (3)
C62 H92 O24 : white amorphous powder; mp 158–161◦ ; [˛]20 D + 80.1◦ (MeOH; ca. 0.1); UV (MeOH) max : 222.5, 279.0 nm; IR (KBr) max : 3480 (OH), 1710 (C C C O), 1640 (C C), 1170 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1243.5894 ([C62 H92 O24 + Na]+ , calcd. 1243.5871). ESI-MS (positive) m/z: 1243.4 [M + Na]+ , 1095.4 [M + Na − 148]+ , 1143.4 [M + Na − 100]+ , 995.4 [M + Na − 148100]+ , 633.3 [M + Na − 610]+ , 489.2 [M + Na − 610-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.4.
Mucronatoside H (4)
C53 H78 O19 : white amorphous powder; mp 153–156◦ ; ◦ [˛]20 D + 72.1 (MeOH; ca. 0.1); UV (MeOH) max : 222.0, 278.5 nm;
685
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
Table 1 – 13 C NMR data of compounds 1–4 (ı in ppm, C5 D5 N)a
Table 1 (Continued)
1
2
3
39.0 (t) 29.4 (t) 78.0 (d) 39.3 (t) 139.4 (s) 119.5 (d) 35.1 (t) 74.3 (s) 45.0 (d) 37.4 (s) 29.9 (t) 68.9 (d) 60.4 (s) 89.3 (s) 34.2 (t) 32.8 (t) 92.6 (s) 9.4 (q) 18.5 (q) 209.6 (s) 27.9 (q)
39.2 (t) 29.2 (t) 77.9 (d) 39.5 (t) 139.3 (s) 119.9 (d) 35.5 (t) 74.2 (s) 44.7 (d) 37.5 (s) 30.1 (t) 70.9 (d) 58.8 (s) 89.0 (s) 34.7 (t) 34.3 (t) 89.1 (s) 11.5 (q) 18.5 (q) 73.2 (d) 17.9 (q)
27.6 (t) 26.6 (t) 74.9 (d) 39.1 (t) 74.8 (s) 136.6 (d) 127.4 (d) 74.0 (s) 36.6 (d) 39.6 (s) 23.7 (t) 75.6 (d) 58.0 (s) 88.2 (s) 33.2 (t) 34.3 (t) 87.8 (s) 12.4 (q) 21.6 (q) 74.5 (d) 15.6 (q)
27.6 (t) 26.6 (t) 74.9 (d) 39.0 (t) 74.8 (s) 136.6 (d) 127.4 (d) 74.0 (s) 36.7 (d) 39.6 (s) 23.7 (t) 75.8 (d) 58.0 (s) 88.2 (s) 33.1 (t) 33.9 (t) 87.5 (s) 12.4 (q) 21.6 (q) 74.5 (d) 15.4 (q)
12-O-Acyl 1 2 3 4 5, 9 6, 8 7
cin 166.7 (s) 120.4 (d) 143.8 (d) 135.2 (s) 128.5 (d) 129.3 (d) 130.5 (d)
cin 166.8 (s) 120.5 (d) 144.1 (d) 135.1 (s) 128.6 (d) 129.4 (d) 130.5 (d)
20-O-Acyl 1 2 3 4 5
tig 166.7 (s) 129.4 (s) 137.7 (d) 14.1 (q) 12.2 (q)
ac 169.8 (s) 21.4 (q)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
4
1 2 3 4 5 6
96.4 (d) 37.0 (t) 78.1 (d) 83.1 (d) 69.3 (d) 18.4 (q)
96.5 (d) 37.1 (t) 78.2 (d) 83.5 (d) 69.2 (d) 18.7 (q)
97.7 (d) 36.9 (t) 78.0 (d) 82.9 (d) 69.3 (d) 18.4 (q)
97.7 (d) 36.8 (t) 77.8 (d) 82.9 (d) 69.1 (d) 18.4 (q)
OCH3
58.9 (q)
59.0 (q)
58.9 (q)
58.8 (q)
d-Cym 1 2 3 4 5 6
100.4 (d) 37.3 (t) 77.7 (d) 83.0 (d) 69.0 (d) 18.6 (q)
100.5 (d) 37.4 (t) 78.1 (d) 83.1 (d) 69.4 (d) 18.6 (q)
100.3 (d) 36.8 (t) 77.9 (d) 82.9 (d) 69.1 (d) 18.6 (q)
100.3 (d) 36.8 (t) 78.1 (d) 83.0 (d) 69.3 (d) 18.5 (q)
OCH3
59.0 (q)
59.1 (q)
58.8 (q)
58.8 (q)
d-Allme 1 2 3 4
104.8 (d) 74.8 (d) 85.9 (d) 83.4 (d)
d-Thv 104.9 (d) 74.9 (d) 86.0 (d) 83.3 (d)
104.8 (d) 74.7 (d) 85.8 (d) 83.1 (d)
2
3
5 6
72.0 (d) 18.7 (q)
72.1 (d) 18.8 (q)
71.9 (d) 18.5 (q)
72.7 (d) 18.5 (q)
OCH3
60.6 (q)
60.7 (q)
60.6 (q)
61.0 (q)
106.2 (d) 75.0 (d) 87.8 (d) 75.8 (d)
4
106.1 (d) 75.9 (d) 78.8 (d) 72.1 (d) 78.2 (d) 63.2 (t)
105.9 (d) 75.8 (d) 78.6 (d) 71.9 (d) 78.2 (d) 63.1 (t)
d-Glc
1 2 3 4 5 6
106.0 (d) 75.8 (d) 78.7 (d) 72.0 (d) 78.1 (d) 63.1 (t)
Multiplicities by DEPT experiments in parentheses; s: quaternary, d: CH, t: CH2 , and q: CH3 C-atoms. allme: 6-deoxy-3-O-methyl-allopyranosyl; cym: cymaropyranosyl; glc: glucopyranosyl; thv: thevetopyranosyl. a 1
H NMR, 13 C NMR, DEPT, 1 H, 1 H COSY, HMQC and HMBC spectra were obtained at 500 and 125 MHz at room temperature, respectively.
IR (KBr) max : 3485 (OH), 1735 (C O), 1711 (C C C O), 1638 (C C), 1165 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1041.4990 ([C53 H78 O19 + Na]+ , calcd. 1041.5030). ESI-MS (positive) m/z: 1041.4 [M + Na]+ , 981.4 [M + Na − 60]+ , 893.4 [M + Na − 148]+ , 875.4 [M + Na − 148-18]+ , 833.4 [M + Na − 148-60]+ , 715.4 [M + Na − 148-18-160]+ , 571.3 [M + Na − 148-18-160-144]+ , 471.2 [M + Na − 570]+ , 327.1 [M + Na − 570-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.5.
d-Cym
1
Acid hydrolysis of 1, 2, 3 and 4
Each solution of 40 mg of 1, 2, 3, and 4 in 10 ml MeOH was treated with 10 ml of 0.1N H2 SO4 , and each mixture was kept at 60 ◦ C for 2 h, then diluted with H2 O (20 ml) and concentrated to 30 ml. The solution was kept for 60 ◦ C for another hour, then neutralized with aq. Satd Ba(OH)2 . The precipitation was then filtered off. The filtrate was concentrated to dryness and chromatographed on a column of silica gel with CHCl3 /MeOH(100:1 → 50:1, v/v) to afford 5 (12.1 mg) from 1, 6 (10.3 mg) from 2, 7 (9.4 mg) from 3, and 8 (11.4 mg) from 4, respectively. The sugar components in each hydrolysate were identified by TLC comparison with authentic samples. Cymarose was identified with the solvent A: CHCl3 /MeOH (9:1); solvent B: Me2 CO/petrol ether (2:3); and solvent C: CH2 Cl2 /EtOH (9:1). Glucose was identified with the solvent CHCl3 /MeOH/H2 O (4:3:1).
2.3.6.
12-O-deacetylmetaplexigenin (5)
C21 H32 O6 : colorless needles (MeOH); ESI-MS m/z 403.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 39.0 (C-1), 31.9 (C-2), 71.4 (C-3), 43.2 (C-4), 140.1 (C-5), 118.6 (C-6), 34.0 (C-7), 74.2 (C-8), 44.8 (C-9), 37.2 (C-10), 29.3 (C-11), 68.8 (C-12), 60.2 (C-13), 89.2 (C-14), 34.9 (C-15), 32.6 (C-16), 92.4 (C-17), 9.2 (C-18), 18.3 (C-19), 209.4 (C-20), 27.7 (C-21). 1 H NMR (C5 D5 N, 500 MHz): ı 3.93 (1H, m, H-3), 5.42 (1H, br s, H-6), 3.98 (1H, dd, J = 11.5, 4.0 Hz, H-12), 2.04 (3H, s, H-18), 1.49 (3H, s, H-19), 2.68 (3H, s, H-21).
686
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
Table 2 – 1 H NMR data of compounds 1–4 (ı in ppm, J in Hz, C5 D5 N)a 1 3 6 7 12 18 19 20 21
2
3.96, m 5.36, br s
3.86, m 5.37, br s
3.94, m 2.00, s 1.42, s
3.89, m 1.93, s 1.41, s 4.44, q (6.0) 1.51, d (6.0)
3
4
4.19, m 5.96, d (10.0) 6.27, d (10.0) 5.38, dd (10.5, 4.0) 2.17, s 1.56, s 5.13, q (6.0) 1.49, d (6.0)
4.19, m 5.96, d (10.5) 6.27, d (10.5) 5.38, dd (10.0, 3.5) 2.12, s 1.58, s 5.02, q (6.0) 1.43, d (6.0)
12-O-Acyl 2 3 5, 9 6, 8 7
cin 6.76, d (16.0) 7.95, d (16.0) 7.67, d (6.5) 7.43, m 7.40, m
cin 6.88, d (15.5) 8.04, d (15.5) 7.75, d (7.0) 7.41, m 7.38, m
20-O-Acyl 2 3 4 5
tig
ac 2.01, s
2.65, s
7.01, q (7.0) 1.51, d (7.0) 1.79, s d-Cym
1 2 3 4 5 6
5.30, d (9.5) 1.73, m; 2.16, m 3.98, m 3.54, dd (9.5, 2.5) 4.24, m 1.38, d (6.0)
5.29, d (9.5) 1.69, m; 2.16, m 3.95, m 3.47, dd (9.5, 2.5) 4.22, m 1.36, d (6.0)
5.19, d (9.5) 1.76, m; 2.22, m 4.02, m 3.45, dd (9.5, 3.0) 4.23, m 1.40, d (6.0)
5.19, d (9.0) 1.72, m; 2.23, m 4.03, m 3.46, dd (9.5, 2.5) 4.17, dq (9.5, 6.5) 1.37, d (6.0)
OCH3
3.62, s
3.60, s
3.63, s
3.63, s
d-Cym 1 2 3 4 5 6
5.12, d (9.5) 1.78, m; 2.17, m 4.03, m 3.88, dd (10.5, 5.0) 4.22, m 1.58, d (6.5)
5.08, d (10.0) 1.75, m; 2.18, m 4.05, m 3.84, m 4.19, m 1.55, d (6.0)
5.12, d (10.0) 1.78, m; 2.24, m 4.08, m 3.91, m 4.21, m 1.58, d (6.0)
5.13, d (10.0) 1.80, m; 2.26, m 4.08, m 3.61, dd (10.0, 2.0) 4.23, dq (9.5, 6.0) 1.62, d (6.0)
OCH3
3.58, s
3.56, s
3.59, s
3.58, s
d-Allme
d-Thv
1 2 3 4 5 6
5.15, d (10.0) 3.92, t (9.0) 3.72, t (8.0) 3.54, dd (9.5, 2.5) 3.81, m 1.76, d (6.0)
5.12, d (10.0) 3.88, t (9.0) 3.69, t (8.0) 3.49, dd (10.0, 2.5) 3.74, dq (8.5, 6.0) 1.73, d (6.0)
5.16, d (10.5) 3.93, m 3.75, m 3.53, dd (9.5, 2.0) 3.79, dq (8.5, 6.0) 1.79, d (6.0)
4.80, d (7.5) 3.95, m 3.66, m 3.64, m 3.76, dq (8.5, 6.0) 1.61, d (6.0)
OCH3
3.96, s
3.92, s
3.97, s
3.93, s
4.69, d (7.5) 4.05, m 4.22, m 4.20, m 4.01, m 4.35, dd (11.0, 5.0) 4.52, d (10.5)
4.73, d (7.5) 4.07, m 4.28, m 4.22, m 4.09, m 4.40, dd (11.0, 5.5) 4.58, dd (11.0, 2.5)
d-Glc 1 2 3 4 5 6””
4.72, d (7.5) 4.04, m 4.25, m 4.21, m 4.03, m 4.37, dd (11.5, 5.5) 4.55, dd (11.5, 2.5)
cym: cymaropyranosyl; glc: glucopyranosyl; thv: thevetopyranosyl. a
Allme: 6-deoxy-3-O-methyl--allopyranosyl.
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
2.3.7.
Sarcostin (6)
C21 H34 O6 : colorless needles (MeOH). ESI-MS m/z 405.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 38.9 (C-1), 31.2 (C-2), 70.7 (C-3), 42.2 (C-4), 139.4 (C-5), 118.5 (C-6), 34.8 (C-7), 73.6 (C-8), 44.1 (C-9), 36.9 (C-10), 29.4 (C-11), 70.3 (C-12), 58.4 (C-13), 88.4 (C-14), 34.1 (C-15), 33.6 (C-16), 88.9 (C-17), 10.6 (C-18), 18.3 (C19), 72.5 (C-20), 17.8 (C-21). 1 H NMR (C5 D5 N, 500 MHz): ı 3.93 (1H, m, H-3), 5.40 (1H, br s, H-6), 3.97 (1H, m, H-12), 1.96 (3H, s, H-18), 1.39 (3H, s, H-19), 4.40 (1H, q, J = 6.0 Hz, H-20), 1.51 (3H, d, J = 6.0 Hz, H-21).
2.3.8. 12-O-cinnamoyl-20-O-tigloyl(20S)-pregn-6-ene3ˇ,5˛,8ˇ,12ˇ,14ˇ,17ˇ,20-heptanol (7) C35 H46 O9 : colorless needles (MeOH). ESI-MS m/z 633.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 27.3 (C-1), 28.7 (C-2), 66.7 (C-3), 41.3 (C-4), 75.3 (C-5), 136.8 (C-6), 127.1 (C-7), 74.1 (C-8), 36.4 (C-9), 39.8 (C-10), 23.8 (C-11), 75.7 (C-12), 58.0 (C13), 88.3 (C-14), 33.2 (C-15), 34.5 (C-16), 87.8 (C-17), 12.4 (C-18), 21.8 (C-19), 74.6 (C-20), 15.6 (C-21), 166.9 (C-1 ), 120.5 (C-2 ), 143.7 (C-3 ), 135.1 (C-4 ), 128.7 (C-5 ,9 ), 129.3 (C-6 ,8 ), 130.5 (C-7 ), 166.9 (C-1 ), 129.4 (C-2 ), 138.1 (C-3 ), 14.2 (C-4 ), 12.3 (C-5 ). 1 H NMR (C5 D5 N, 500 MHz): ı 4.32 (1H, m, H-3), 5.98 (1H, d, J = 10.0 Hz, H-6), 6.23 (1H, d, J = 10.0 Hz, H-7), 5.39 (1H, dd, J = 10.0, 4.0 Hz, H-12), 2.12 (3H, s, H-18), 1.60 (3H, s, H19), 5.12 (1H, q, J = 6.0 Hz, H-20), 1.49 (3H, d, J = 6.0 Hz, H-21), 6.75 (1H, d, J = 16.0 Hz, H-2 ), 7.92 (1H, d, J = 16.0 Hz, H-3 ), 7.64 (2H, dd, J = 7.0, 1.5 Hz, H-5 , 9 ), 7.39 (2H, dd, J = 7.0, 6.5 Hz, H-6 , 8 ), 7.36 (1H, ddd, J = 7.0, 6.5, 1.5 Hz, H-7 ), 6.96 (1H, q, J = 6.5 Hz, H-3 ), 1.46 (3H, d, J = 6.5 Hz, H-4 ), 1.77 (3H, s, H5 ). 13 C NMR (DMSO-d , 125 MHz): 26.3 (C-1), 27.4 (C-2), 66.0 6 (C-3), 40.1(C-4), 74.3 (C-5), 135.7 (C-6), 126.1 (C-7), 73.0 (C-8), 34.8 (C-9), 38.4 (C-10), 22.6 (C-11), 74.8 (C-12), 57.1 (C-13), 87.2 (C-14), 32.2 (C-15), 33.4 (C-16), 87.1 (C-17), 11.7 (C-18), 21.3 (C-19), 73.6 (C-20), 15.2 (C-21), 165.7 (C-1 ), 119.3 (C-2 ), 143.5 (C-3 ), 134.1 (C-4 ), 128.4 (C-5 ,9 ), 129.0 (C-6 ,8 ), 130.1 (C-7 ), 166.1 (C-1 ), 128.4 (C-2 ), 137.7 (C-3 ), 14.3 (C-4 ), 12.1 (C-5 ). 1 H NMR (DMSO-d , 500 MHz): ı 3.97 (1H, m, H-3), 5.38 (1H, 6 d, J = 10.0 Hz, H-6), 5.65 (1H, d, J = 10.0 Hz, H-7), 4.72 (1H, dd, J = 10.0, 4.5 Hz, H-12), 1.46 (3H, s, H-18), 0.92 (3H, s, H-19), 4.45 (1H, q, J = 6.0 Hz, H-20), 1.09 (3H, d, J = 6.0 Hz, H-21), 6.21 (1H, d, J = 16.0 Hz, H-2 ), 7.42 (1H, d, J = 16.0 Hz, H-3 ), 7.60 (2H, dd, J = 7.5, 1.5 Hz, H-5 ,9 ), 7.39 (2H, m, H-6 ,8 ), 7.38 (1H, m, H-7 ), 6.75 (1H, q, J = 7.0 Hz, H-3 ), 1.61 (3H, d, J = 7.0 Hz, H-4 ), 1.53 (3H, s, H-5 ).
2.3.9. 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene3ˇ,5˛,8ˇ,12ˇ,14ˇ,17ˇ,20-heptanol (8) C32 H42 O9 : colorless needles (MeOH). ESI-MS m/z 593.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 27.5 (C-1), 28.7 (C-2), 66.6 (C-3), 41.3 (C-4), 75.1 (C-5), 136.5 (C-6), 127.3 (C-7), 74.2 (C-8), 36.5 (C-9), 39.7 (C-10), 23.7 (C-11), 75.7 (C-12), 58.1 (C-13), 88.3 (C-14), 33.1 (C-15), 34.1 (C-16), 87.7 (C-17), 12.5 (C-18), 21.7 (C19), 74.6 (C-20), 15.6 (C-21), 166.9 (C-1 ), 120.5 (C-2 ), 143.8 (C-3 ), 135.1 (C-4 ), 128.6 (C-5 ,9 ), 129.1 (C-6 ,8 ), 130.5 (C-7 ), 169.7 (C1 ), 21.5 (C-2 ). 1 H NMR (C5 D5 N, 500 MHz): ı 4.26 (1H, m, H-3), 5.94 (1H, d, J = 10.5 Hz, H-6), 6.22 (1H, d, J = 10.5 Hz, H-7), 5.36 (1H, dd, J = 10.0, 3.5 Hz, H-12), 2.12 (3H, s, H-18), 1.59 (3H, s, H19), 5.06 (1H, q, J = 6.0 Hz, H-20), 1.47 (3H, d, J = 6.0 Hz, H-21), 6.85 (1H, d, J = 16.0 Hz, H-2 ), 7.97 (1H, d, J = 16.0 Hz, H-3 ), 7.70 (2H, d,
687
J = 7.0 Hz, H-5 ,9 ), 7.38 (2H, m, H-6 ,8 ), 7.35 (1H, m, H-7 ), 2.02 (1H, s, H-2 ).
2.4.
Splenocyte proliferation assay
Single cell suspensions were prepared as previously described [9]. Splenocytes were seeded into four wells of a 96-well flatbottom microtiter plate (Nunc) at a cell density of 1 × 107 per ml in 100 l of complete medium, into which 100 l of 1–4 and CsA (0.01–10 g/ml) were added, followed by Con A (final concentration 5 mg l−1 ), LPS (final concentration 10 mg l−1 ), or medium. The plate was incubated at 37 ◦ C under a humidified atmosphere of 5% CO2 . After 44 h, 50 l of MTT solution (2 g l−1 ) was added to each of the wells, which were incubated for 4 h. The microtiter plates were centrifuged (1400 × g, 5 min), and the untransformed MTT was removed carefully by pipetting. Next, 200 l of a Me2 SO working solution (192 l Me2 SO with 8 l HCl 1 mol l−1 ) was added to each well, and the absorbance (A) was evaluated after 15 min in an ELISA reader at 570 nm with a 630 nm reference.
3.
Results and discussion
The EtOH extract of the stems of S. mucronata was extracted with CHCl3 . The CHCl3 soluble portion was subsequently separated using silica, reversed-phase silica gel column chromatography and semi-prep HPLC to yielded four compounds (1–4). These compounds showed positive Libermann–Buchard and Keller–Kiliani reactions, indicating the presence of a steroidal glycoside with a 2-deoxysugar moiety. Each of the isolates was subjected to detailed spectroscopic analysis to establish their chemical structures. Mucronatoside E (1) was isolated as an amorphous powder. Based on the HRESI-MS data, the molecular formula of 1 was determined to be C48 H78 O21 (1013.4923 [M + Na]+ , calcd. 1013.4928). Mild acid hydrolysis of 1 afforded an aglycone (5), cymarose, d-glucose, and an unidentified sugar (on TLC). The aglycone was identified as 12-O-deacetylmetaplexigenin, by comparison of its spectroscopic data to those in the literature [7]. The NMR (1 H NMR, 13 C NMR, DEPT, HMQC, and HMBC) spectral data of compound 1 showed that it contained four anomeric carbon signals at ı 96.4, 100.4, 104.8, and 106.0, correlating with anomeric protons at ı 5.30 (d, J = 9.5 Hz), 5.12 (d, J = 9.5 Hz), 5.15 (d, J = 10.0 Hz), and 4.72 (d, J = 7.5 Hz), respectively, which indicated that there were four sugar units in the compound. The sugar moieties of 1 were identical to those of stemucronatoside C, by comparison of their 1 H NMR and 13 C NMR data and TLC evidence with those in the literature [7]. The sugar sequence of compound 1 was confirmed by the HMBC spectrum, which showed long-range correlations between (i) H-1 of the -d-cymaropyranose (ıH 5.30, 1H, d, J = 9.5 Hz) and C-3 of the aglycone (ıC 78.0); (ii) H-1 of the -d-cymaropyranose (ıH 5.12, 1H, d, J = 9.5 Hz) and C-4 of the -d-cymaropyranose (ıC 83.1); (iii) H-1 of the 6-deoxy-3-O-methyl--d-allopyranose (abbreviated as allme) (ıH 5.15, 1H, d, J = 10.0 Hz) and C-4 of the -d-cymaropyranose (ıC 83.0); and (iv) H-1 of the ˇ-d-glucopyranose (ıH 4.72, 1H, d, J = 7.5 Hz) and C-4 of the 6-deoxy-3-O-methyl--dallopyranose (ıC 83.4). Thus, the sequence and linkage sites
688
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
of the sugar units were established as glc-(1 → 4)-allme(1 → 4)-cym-(1 → 4)-cym-(1 → 3)-aglycone. This assignment was consistent with the ESI-MS data, which showed the quasi-molecular ion at m/z 1013.4 [M + Na]+ , and other prominent fragments at m/z 851.4 [M + Na − glc]+ , 691.3 [M + Na − glc-allme]+ , 547.2 [M + Na − glc-allme-cym]+ , and 403.1, which corresponded to the aglycone moiety. In the 13 C NMR spectrum of 1, glycosidation shifts were observed at C-2, C-3, and C-4 by −2.5, +6.6 and −3.9, respectively, when compared with 13 C chemical shifts of 5, indicating that the sugar chain was linked to the C-3 hydroxyl group of the aglycone. Thus, the structure of 1 was established as 12-O-deacetylmetaplexigenin 3-O--d-glucopyranosyl(1 → 4)-6-deoxy-3-O-methyl--d-allopyranosyl-(1 → 4)--dcymaropyranosyl-(1 → 4)--d-cymaropyranoside. Mucronatoside F (2) possessed the molecular formula C48 H80 O21 (1015.5103 [M + Na]+ , calcd. 1015.5084) on the basis of the HRESI-MS. Mild acid hydrolysis of 2 afforded an aglycone (6) and a sugar mixture. 6 was identical to sarcostin by comparison of its spectroscopic data with those in the literature [10]. In 1975, the absolute configuration of 6 was confirmed by the X-ray analysis of its 3-O-p-bromobenzoate which showed S-configuration at C-20 by Hayashi and Mitsuhashi et al. [11]. Moreover, in the 20-hydroxy-C/d-cis-pregnane type steroids the free rotation of the side-chain is considered to be restricted by steric hindrance among the C-18 methyl, C-21 methyl and 20-hydroxyl groups on the basis of a CPK model [12]. In compound 6, the C-17 OH further restricted the rotate of the side-chain. Thus, the NOESY evidence of (i) H-12 at ı 3.97 and H-20 at ı 4.40; (ii) H-16␣ at ı 1.90 and H-20 at ı 4.40; and (iii) H-16 at ı 1.85 and H-21 at ı 1.51 can further confirm the 20-S configuration. The 1 H and 13 C NMR spectral data for the aglycone moiety of 2 were similar to those of sarcostin (6) with the major difference being the absence of signals of an OH group at C-3. The only other difference in the 13 C NMR data between 2 and 6 occurred for C-2, C-3, and C-4, by −2.0, +7.2, and −2.7, respectively. Therefore, the sugar moiety was linked to the C-3 hydroxyl group of the aglycone. The NMR assignments of 2 were made unambiguously on the basis of HMQC, HMBC and 1 H, and 1 H COSY experiments; by comparison to those of 1, 2 was determined to have the same sugar sequence in the oligosaccharide moiety. Thus, compound 2 was established as sarcostin 3-O--d-glucopyranosyl(1 → 4)-6-deoxy-3-O-methyl--d-allopyranosyl-(1 → 4)--dcymaropyranosyl-(1 → 4)--d-cymaropyranoside. Mucronatoside G (3) possessed the molecular formula C62 H92 O24 (1243.5894 [M + Na]+ , calcd. 1243.5871) on the basis of the HRESI-MS. Mild acid hydrolysis of 3 afforded an aglycone (7) and a sugar mixture. The molecular formula of 7 was deduced as C35 H46 O9 by the quasi-molecular ion peak at m/z 633.1 [M + Na]+ in the ESI-MS spectra and by its 13 C NMR data. 7 was identical to 12-O-cinnamoyl-20-O-tigloyl(20S)pregn-6-ene-3,5␣,8,12,14,17,20-heptanol by comparison of its spectroscopic data with those in the literature [13], but the C-1 chemical shifts of the cinnamoyl group and the tigloyl group were both at 166.9 ppm. In order to locate the cinnamoyl group and tigloyl group in 7, a HMBC experiment (in DMSO-d6 ) had to be carried out. In the HMBC spectrum, the following long-range correlations were observed between (i) H-12 (ıH 4.72) and C-1 of the cinnamoyl (ıC 165.7); and (ii)
H-20 (ıH 4.45) and C-1 of the tigloyl (ıC 166.1). Thus, 7 was determined to be 12-O-cinnamoyl-20-O-tigloyl (20S)-pregn-6ene-3,5␣,8,12,14,17,20-heptanol. The 1 H and 13 C NMR spectral data for the aglycone moiety of compound 3 were similar to those of 7 with the major difference being the absence of signals for an OH group at C-3. The only other difference in 13 C NMR spectral data between 3 and 7 occurred for C-atoms C-2, C-3, and C-4, by −2.1, +8.2, and −2.2, respectively. The 1 H and 13 C NMR signals due to the sugar moieties of 3 were also superimposable onto those of 1. Consequently, the structure of 3 was established as 12-O-cinnamoyl-20-Otigloyl(20S)-pregn-6-ene-3,5␣,8,12,14,17,20-heptanol 3O--d-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl--d-allopyranosyl-(1 → 4)--d-cymaropyranosyl-(1 → 4)--dcymaropyranoside. Mucronatoside H (4) had the molecular formula C53 H78 O19 (1041.4990 [M + Na]+ , calcd. 1041.5030) on the basis of the HRESI-MS data. Mild acid hydrolysis of 4 afforded an aglycone (8), cymarose, and an unidentified sugar (on TLC). The molecular formula of 8 was deduced as C32 H42 O9 by the quasimolecular ion peak at m/z 593.1 [M + Na]+ in the ESI-MS spectra and its 13 C NMR data. The spectral analysis of 8 revealed a distinct similarity to 7. In comparison to 7, 8 showed the absence of the tigloyl group, but suggested the presence of an acetyl group. In the HMBC spectrum of 8, the following long-range correlations were observed: (i) between H-12 (ıH 5.36) and C-1 of the cinnamoyl (ıC 166.9), and (ii) between H-20 (ıH 5.06) and C-1 of the acetyl (ıC 169.7). Thus, 8 was determined to be 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6ene -3,5␣,8,12,14,17,20-heptanol. The NMR (1 H NMR, 13 C NMR, DEPT, HMQC, and HMBC) spectral data of compound 4 showed that it contained three anomeric carbon signals at ı 97.7, 100.3, and 106.2, correlating with anomeric protons at ı 5.19 (d, J = 9.0 Hz), 5.13 (d, J = 10.0 Hz), and 4.80 (d, J = 7.5 Hz), respectively, which indicated that there were three sugar units in compound 1. The sugar moieties of 4 were identical to those of stemucronatoside B, deduced by comparing of their 1 H and 13 C NMR data and TLC data to those in the literature [7]. The sequence of sugars in 4 was confirmed by the HMBC spectrum, which showed distinct cross-peaks of correlation between (i) H-1 of the -d-cymaropyranose (ıH 5.19, 1H, d, J = 9.0 Hz) and C-3 (ıC 74.9); (ii) H-1 of the -d-cymaropyranose (ıH 5.13, 1H, d, J = 10.0 Hz) and C-4 of the -d-cymaropyranose (ıC 82.9); and (iii) H-1 of the -d-thevetopyranose (ıH 4.80, 1H, d, J = 7.5 Hz) and C-4 of the -d-cymaropyranose (ıC 83.0). The glycosidation shifts of the carbon signals of 4 were observed at C-2, C-3, and C-4, by −2.1, +8.3, and −2.3, respectively, compared to 8, indicating the sugar chain linked to the C-3 hydroxyl group of the aglycone. Consequently, the structure of 4 was established as 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene3,5␣,8, 12,14,17,20-heptanol 3-O--d-thevetopyranosyl(1 → 4)--d-cymaropyranosyl-(1 → 4)--d-cymaropyranoside. Comparing the 13 C-NMR data of compounds 1–4 (Table 1), the chemical shifts of the C-1 and C-9 of compounds 3 and 4 are ca. 12 and 9 ppm high-field shifted than that of compounds 1 and 2, respectively. Those phenomena could be relate to that (i) compounds 3 and 4 had the 5␣-OH group, which make the ␥-position carbons C-1 and C-9 up-field, and (ii) C-1 and C-9 in compounds 3 and 4 were in the shielding regions of the double bond between C-6 and C-7.
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Fig. 2 – Effect of compounds 1–4 on Con A-stimulated splenocyte proliferation in vitro. Splenocytes were cultured with the various concentrations of compounds 1–4 or CsA and Con A (5 g/ml) in RPMI 1640 medium for 48 h. Cellular proliferation was measured by the MTT method as described in the text and shown as a stimulation index. The values are presented as means ± S.E. (n = 4). Significant differences compared to 0 g/ml are designated as a P < 0.05, b P < 0.01, and c P < 0.001. CsA: cyclosporin A (positive drug).
Fig. 3 – Effect of compounds 1–4 on LPS-stimulated splenocyte proliferation in vitro. Splenocytes were cultured with the various concentrations of compounds 1–4 or CsA and LPS (10 g/ml) in RPMI 1640 medium for 48 h. Cellular proliferation was measured by the MTT method as described in the text and shown as a stimulation index. The values are presented as means ± S.E. (n = 4). Significant differences compared to 0 g/ml are designated as a P < 0.05, b P < 0.01, and c P < 0.001. CsA: cyclosporin A (positive drug).
The effects of 1–4 on mitogen-stimulated mice splenocyte proliferation in vitro are shown in Figs. 2 and 3. Compounds 2 and 4 significantly inhibited Con A- and LPS-stimulated mice splenocyte proliferation in a dose-dependent manner. However, compound 1 and 3 significantly enhanced Con A- and LPS-induced mice splenocyte proliferation at suitable concentrations. There were no significant differences in inhibitory effect on mitogen-stimulated splenocyte proliferation among CsA, compounds 2 and 4 at the concentration of 1 g/ml. CsA showed cytotoxic activity mice splenocyte at the concentration of 10 g/ml, while compounds 2 and 4 had no influences on mice splenocyte growth at the same concentration. The inhibitory effect compounds 2 and 4 on mitogen-stimulated splenocyte proliferation at the concentration of 10 g/ml was more significant than that of CsA at the concentration of 1 g/ml (P < 0.05).
Acknowledgements We gratefully acknowledge the Zhejiang Provincial Natural Science Foundation of China (Grant-in Aid No. M303749) and the Zhejiang Provincial Science and Technology Council (Grant-in Aid No. 2004F13G1360002) for financial support.
references
[1] Luo SQ, Lin LZ, Cordell GA, Xue L, Johnson ME. Polyoxypregnanes from Marsdenia tenacissima. Phytochemistry 1993;34:1615–20. [2] Hayashi K, Wada K, Mitsuhashi H, Bando H, Takase M. Antitumor active glycosides from Condurango Cortex. Chem Pharm Bull 1980;28:1954–8.
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[3] Mu QZ, Lu JL, Zhou QL. The antiepilepsy constituents of Cynanchum otophyllum Schneid: the structures of otophylosides A and B. Sci Sin Ser B 1985;(8):724–30. [4] Zhang YE, Yuan JL, Ding WP. Studies on the antifertility constituents of Marsdenia oreophila. Acta Pharmaceutica Sinica 1994;29:281–4. [5] Zhang RS, Ye YP, Li XY, Zhang XY. Steroidal glycosides from the stems of Stephanotis mucronata (Blanco) Merr. (Asclepiadaceae). Acta Chim Sin 2003;61:1991–6. [6] Zhang RS, Ye YP, Li XY, Zhang XY. Oxypregnane-oligoglycosides from the stems of Stephanotis mucronata (Asclepiadaceae). Chin Chem Lett 2005;16:483–6. [7] Ye YP, Li XY, Sun HX, Chen FY, Pan YJ. Immunomodulating steroidal glycosides from the roots of Stephanotis mucronata. Helv Chim Acta 2004;87:2378–84. [8] Ye YP, Sun HX, Li XY, Chen FY, Qin F, Pan YJ. Four new C-21 steroidal glycosides from the roots of Stephanotis mucronata and their immunological activities. Steroids 2005;70:791–7.
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[9] Sun HX, Qin F, Pan YJ. In vitro and in vivo immunosuppressive activity of Spica Prunellae ethanol extract on the immune responses in mice. J Ethnopharmacol 2005;101:31–6. [10] Warshina T, Noro T. Steroidal glycosides from Cynanchum caudatum. Phytochemistry 1995;39:199–204. [11] Hayashi K, Mitsuhashi H. Studies on the constituents of Asclepiadaceae Plants. XXXIV. Chemistry of sarcostin. Chem Pharm Bull 1975;23:1845–51. [12] Kimura M, Hayashi K, Narita H, Mitsuhashi H. Studies on the constituents of Asclepiadaceae plants. LI. Oxidation at the 18-methyl group of C/d-cis-pregnane type steroids and 13 C-nuclear magnetic resonance spectra of 18-oxygenated pregnanes and related compounds. Chem Pharm Bull 1982;30:3932–41. [13] Yoshikawa K, Okada N, Kan Y, Arihara S. Steroidal glycosides from the fresh stem of Stephanotis lutchuensis var. Japonica (Asclepiadaceae). Chemical structures of stephanosides K–Q. Chem Pharm Bull 1996;44:2243–8.
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Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata Xiaoyu Li a,b , Hongxiang Sun c , Yiping Ye b , Fengyang Chen b , Jue Tu c , Yuanjiang Pan a,∗ a b c
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated
Received 15 September 2005
from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double
Received in revised form 29 March
bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic
2006
data and chemical evidence. These isolated compounds were assayed for their immunolog-
Accepted 17 April 2006
ical activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced
Published on line 21 June 2006
proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing
Keywords:
activities. © 2006 Elsevier Inc. All rights reserved.
Stephanotis mucronata Asclepiadaceae Immunomodulating activities
1.
Introduction
Immunosuppressive agents are the mainstay treatment for patients that have received organ grafts and are becoming increasingly important in the treatment of autoimmune diseases. There are, however, many problems with both the concept and reality of long-term immunosuppression as a therapeutic modality, both in terms of the nonspecific toxicity of the drugs that are currently available and the increased risk of infections and tumors arising from global suppression of the immune system. The immunosuppressant with less side effects is still a challenge to the medical system. C-21 steroidal glycosides have attracted much attention for their antitumor, antiepilepsy, and antifertility activities, and many such compounds have been isolated from plants, especially from those of the Asclepiadaceae family [1–4]. The dried roots of Stephanotis mucronata (Blanco) Merr. (Asclepi-
∗
adaceae) are used for the treatment of rheumatoid arthritis and rheumatic aches in Chinese folk medicine. We have earlier reported the isolation and structural elucidation of 12 pregnane glycosides: mucronatosides A–D, and stephanoside E from the stems of S. mucronata [5,6], and stemucronatosides A–G from the roots of S. mucronata [7,8]. We have also reported the immunomodulating activity of stemucronatosides A–G [7,8]. Among those compounds, stemucronatosides A–C, and F showed immunomodulating activities, while stemucronatosides D, E, and G showed immunosuppressive activities in a dose-dependent manner. In this paper, the chemical studies on the CHCl3 soluble extract from the stems of this plant were further undertaken, leading to four new immunomodulating C-21 steroidal glycosides: mucronatosides E–H (1–4) (Fig. 1). These results might be responsible, at least in part, for the treatment of rheumatoid arthritis and rheumatic aches in folk.
Corresponding author. Tel.: +86 571 879 51264; fax: +86 571 879 51264. E-mail address: [email protected] (Y. Pan). 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.04.007
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2.2.
Plant material
The stems of S. mucronata were obtained from Yueqing, Zhejiang province, China. A voucher specimen (No. 200208) was identified by Prof. Zhang Zhi-Guo and deposited in the Laboratory of Natural Products Chemistry, Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, China.
2.3.
Extraction and isolation
The dried stems of S. mucronata (6.0 kg) were ground and extracted three times with 95% EtOH under reflux for 2 h. The extracts were evaporated in vacuo to give the EtOH extract (570 g). The ethanolic extract was partitioned with CHCl3 under reflux. Concentration of the CHCl3 extract yielded a yellow residue (223.5 g). The CHCl3 extract was partitioned with hexane under reflux. The hexane insoluble portion corresponded to a crude glycoside (193.5 g). The crude glycoside was subjected to CC (silica gel, gradient CHCl3 /MeOH 40:1 → 5:1, v/v) to give 8 main fractions. Fr. 3 (6.2 g) was subjected to CC (Rp-18, MeOH/H2 O 1:1 → 2:1, v/v; then HPLC, MeOH/H2 O 70:30, v/v) to give 4 (135 mg). Fr. 4 (9.4 g) was subjected to CC (Rp-18, MeOH/H2 O 1:1 → 2:1, v/v; then HPLC, MeOH/H2 O 67:33, v/v) to give 1 (155 mg), 2 (142 mg), and 3 (86 mg).
2.3.1.
Fig. 1 – The chemical structures of compounds 1–4.
2.
Experimental
2.1.
General methods
Melting points were determined on an MRK Prism Scope instrument and are uncorrected. Optical rotations were measured in MeOH with a Rudolph Research Autopol IV polarimeter. UV spectra were performed on a TU-1800PC spectrometer. IR spectra were recorded in a KBr pellet on a Perkin-Elmer 577 spectrometer. 1 H NMR, 13 C NMR, DEPT, 1 H, 1 H COSY, HMQC, and HMBC spectra were recorded at 500 MHz for 1 H, and at 125 MHz for 13 C, with a Bruker DRX 500 instrument in pyridined5 solution. HRESI-MS spectra were recorded on a Bruker Daltonics FTMS APEX III mass spectrometer. ESI-MS spectra were recorded on a Bruker Esquire 3000plus mass spectrometer. TLC was performed on precoated Kieselgel 60 F254 plates and Rp C18 (Merck), and the detection was achieved by spraying with 10% H2 SO4 followed by heating. Separation and purification were performed by column chromatography on silica gel (200–300 mesh, Qingdao), Rp C18 (Merck), and on semi-prep HPLC using an Agilent 1100 instrument (Zorbax column 9 mm × 250 mm, DAD).
Mucronatoside E (1)
C48 H78 O21 : white amorphous powder; mp 155–158◦ ; ◦ (MeOH; ca. 0.1); UV (MeOH) [˛]20 max : 207.0 nm; D + 34.1 IR (KBr) max : 3510 (OH), 1701 (C O), 1640 (C C), 1165 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1013.4923 ([C48 H78 O21 + Na]+ , calcd. 1013.4928). ESI-MS (positive) m/z: 1013.4 [M + Na]+ , 851.4 [M + Na − 162]+ , 691.3 [M + Na − 162-160]+ , 547.2 [M + Na − 162-160-144]+ , 403.1 [M + Na − 162 − 160-144-144]+ , 633.2 [M + Na − 380]+ , 489.2 [M + Na − 380-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.2.
Mucronatoside F (2)
C48 H80 O21 : white amorphous powder; mp 165–167◦ ; [˛]20 D + 18.0◦ (MeOH; ca. 0.1); UV (MeOH) max : 207.0 nm; IR (KBr) max : 3480 (OH), 1640 (C C), 1165 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1015.5103 ([C48 H80 O21 + Na]+ , calcd. 1015.5084). ESIMS (positive) m/z: 1015.4 [M + Na]+ , 853.4 [M + Na − 162]+ , 693.3 [M + Na − 162-160]+ , 549.3 [M + Na − 162-160-144]+ , 633.2 [M + Na − 382]+ , 489.1 [M + Na − 382-144]+ , 345.1 [M + Na − 382144-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.3.
Mucronatoside G (3)
C62 H92 O24 : white amorphous powder; mp 158–161◦ ; [˛]20 D + 80.1◦ (MeOH; ca. 0.1); UV (MeOH) max : 222.5, 279.0 nm; IR (KBr) max : 3480 (OH), 1710 (C C C O), 1640 (C C), 1170 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1243.5894 ([C62 H92 O24 + Na]+ , calcd. 1243.5871). ESI-MS (positive) m/z: 1243.4 [M + Na]+ , 1095.4 [M + Na − 148]+ , 1143.4 [M + Na − 100]+ , 995.4 [M + Na − 148100]+ , 633.3 [M + Na − 610]+ , 489.2 [M + Na − 610-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.4.
Mucronatoside H (4)
C53 H78 O19 : white amorphous powder; mp 153–156◦ ; ◦ [˛]20 D + 72.1 (MeOH; ca. 0.1); UV (MeOH) max : 222.0, 278.5 nm;
685
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
Table 1 – 13 C NMR data of compounds 1–4 (ı in ppm, C5 D5 N)a
Table 1 (Continued)
1
2
3
39.0 (t) 29.4 (t) 78.0 (d) 39.3 (t) 139.4 (s) 119.5 (d) 35.1 (t) 74.3 (s) 45.0 (d) 37.4 (s) 29.9 (t) 68.9 (d) 60.4 (s) 89.3 (s) 34.2 (t) 32.8 (t) 92.6 (s) 9.4 (q) 18.5 (q) 209.6 (s) 27.9 (q)
39.2 (t) 29.2 (t) 77.9 (d) 39.5 (t) 139.3 (s) 119.9 (d) 35.5 (t) 74.2 (s) 44.7 (d) 37.5 (s) 30.1 (t) 70.9 (d) 58.8 (s) 89.0 (s) 34.7 (t) 34.3 (t) 89.1 (s) 11.5 (q) 18.5 (q) 73.2 (d) 17.9 (q)
27.6 (t) 26.6 (t) 74.9 (d) 39.1 (t) 74.8 (s) 136.6 (d) 127.4 (d) 74.0 (s) 36.6 (d) 39.6 (s) 23.7 (t) 75.6 (d) 58.0 (s) 88.2 (s) 33.2 (t) 34.3 (t) 87.8 (s) 12.4 (q) 21.6 (q) 74.5 (d) 15.6 (q)
27.6 (t) 26.6 (t) 74.9 (d) 39.0 (t) 74.8 (s) 136.6 (d) 127.4 (d) 74.0 (s) 36.7 (d) 39.6 (s) 23.7 (t) 75.8 (d) 58.0 (s) 88.2 (s) 33.1 (t) 33.9 (t) 87.5 (s) 12.4 (q) 21.6 (q) 74.5 (d) 15.4 (q)
12-O-Acyl 1 2 3 4 5, 9 6, 8 7
cin 166.7 (s) 120.4 (d) 143.8 (d) 135.2 (s) 128.5 (d) 129.3 (d) 130.5 (d)
cin 166.8 (s) 120.5 (d) 144.1 (d) 135.1 (s) 128.6 (d) 129.4 (d) 130.5 (d)
20-O-Acyl 1 2 3 4 5
tig 166.7 (s) 129.4 (s) 137.7 (d) 14.1 (q) 12.2 (q)
ac 169.8 (s) 21.4 (q)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
4
1 2 3 4 5 6
96.4 (d) 37.0 (t) 78.1 (d) 83.1 (d) 69.3 (d) 18.4 (q)
96.5 (d) 37.1 (t) 78.2 (d) 83.5 (d) 69.2 (d) 18.7 (q)
97.7 (d) 36.9 (t) 78.0 (d) 82.9 (d) 69.3 (d) 18.4 (q)
97.7 (d) 36.8 (t) 77.8 (d) 82.9 (d) 69.1 (d) 18.4 (q)
OCH3
58.9 (q)
59.0 (q)
58.9 (q)
58.8 (q)
d-Cym 1 2 3 4 5 6
100.4 (d) 37.3 (t) 77.7 (d) 83.0 (d) 69.0 (d) 18.6 (q)
100.5 (d) 37.4 (t) 78.1 (d) 83.1 (d) 69.4 (d) 18.6 (q)
100.3 (d) 36.8 (t) 77.9 (d) 82.9 (d) 69.1 (d) 18.6 (q)
100.3 (d) 36.8 (t) 78.1 (d) 83.0 (d) 69.3 (d) 18.5 (q)
OCH3
59.0 (q)
59.1 (q)
58.8 (q)
58.8 (q)
d-Allme 1 2 3 4
104.8 (d) 74.8 (d) 85.9 (d) 83.4 (d)
d-Thv 104.9 (d) 74.9 (d) 86.0 (d) 83.3 (d)
104.8 (d) 74.7 (d) 85.8 (d) 83.1 (d)
2
3
5 6
72.0 (d) 18.7 (q)
72.1 (d) 18.8 (q)
71.9 (d) 18.5 (q)
72.7 (d) 18.5 (q)
OCH3
60.6 (q)
60.7 (q)
60.6 (q)
61.0 (q)
106.2 (d) 75.0 (d) 87.8 (d) 75.8 (d)
4
106.1 (d) 75.9 (d) 78.8 (d) 72.1 (d) 78.2 (d) 63.2 (t)
105.9 (d) 75.8 (d) 78.6 (d) 71.9 (d) 78.2 (d) 63.1 (t)
d-Glc
1 2 3 4 5 6
106.0 (d) 75.8 (d) 78.7 (d) 72.0 (d) 78.1 (d) 63.1 (t)
Multiplicities by DEPT experiments in parentheses; s: quaternary, d: CH, t: CH2 , and q: CH3 C-atoms. allme: 6-deoxy-3-O-methyl-allopyranosyl; cym: cymaropyranosyl; glc: glucopyranosyl; thv: thevetopyranosyl. a 1
H NMR, 13 C NMR, DEPT, 1 H, 1 H COSY, HMQC and HMBC spectra were obtained at 500 and 125 MHz at room temperature, respectively.
IR (KBr) max : 3485 (OH), 1735 (C O), 1711 (C C C O), 1638 (C C), 1165 (C O), 1080 (C O C) cm−1 . HRESI-MS: 1041.4990 ([C53 H78 O19 + Na]+ , calcd. 1041.5030). ESI-MS (positive) m/z: 1041.4 [M + Na]+ , 981.4 [M + Na − 60]+ , 893.4 [M + Na − 148]+ , 875.4 [M + Na − 148-18]+ , 833.4 [M + Na − 148-60]+ , 715.4 [M + Na − 148-18-160]+ , 571.3 [M + Na − 148-18-160-144]+ , 471.2 [M + Na − 570]+ , 327.1 [M + Na − 570-144]+ . For 1 H and 13 C NMR data, see Tables 1 and 2.
2.3.5.
d-Cym
1
Acid hydrolysis of 1, 2, 3 and 4
Each solution of 40 mg of 1, 2, 3, and 4 in 10 ml MeOH was treated with 10 ml of 0.1N H2 SO4 , and each mixture was kept at 60 ◦ C for 2 h, then diluted with H2 O (20 ml) and concentrated to 30 ml. The solution was kept for 60 ◦ C for another hour, then neutralized with aq. Satd Ba(OH)2 . The precipitation was then filtered off. The filtrate was concentrated to dryness and chromatographed on a column of silica gel with CHCl3 /MeOH(100:1 → 50:1, v/v) to afford 5 (12.1 mg) from 1, 6 (10.3 mg) from 2, 7 (9.4 mg) from 3, and 8 (11.4 mg) from 4, respectively. The sugar components in each hydrolysate were identified by TLC comparison with authentic samples. Cymarose was identified with the solvent A: CHCl3 /MeOH (9:1); solvent B: Me2 CO/petrol ether (2:3); and solvent C: CH2 Cl2 /EtOH (9:1). Glucose was identified with the solvent CHCl3 /MeOH/H2 O (4:3:1).
2.3.6.
12-O-deacetylmetaplexigenin (5)
C21 H32 O6 : colorless needles (MeOH); ESI-MS m/z 403.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 39.0 (C-1), 31.9 (C-2), 71.4 (C-3), 43.2 (C-4), 140.1 (C-5), 118.6 (C-6), 34.0 (C-7), 74.2 (C-8), 44.8 (C-9), 37.2 (C-10), 29.3 (C-11), 68.8 (C-12), 60.2 (C-13), 89.2 (C-14), 34.9 (C-15), 32.6 (C-16), 92.4 (C-17), 9.2 (C-18), 18.3 (C-19), 209.4 (C-20), 27.7 (C-21). 1 H NMR (C5 D5 N, 500 MHz): ı 3.93 (1H, m, H-3), 5.42 (1H, br s, H-6), 3.98 (1H, dd, J = 11.5, 4.0 Hz, H-12), 2.04 (3H, s, H-18), 1.49 (3H, s, H-19), 2.68 (3H, s, H-21).
686
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
Table 2 – 1 H NMR data of compounds 1–4 (ı in ppm, J in Hz, C5 D5 N)a 1 3 6 7 12 18 19 20 21
2
3.96, m 5.36, br s
3.86, m 5.37, br s
3.94, m 2.00, s 1.42, s
3.89, m 1.93, s 1.41, s 4.44, q (6.0) 1.51, d (6.0)
3
4
4.19, m 5.96, d (10.0) 6.27, d (10.0) 5.38, dd (10.5, 4.0) 2.17, s 1.56, s 5.13, q (6.0) 1.49, d (6.0)
4.19, m 5.96, d (10.5) 6.27, d (10.5) 5.38, dd (10.0, 3.5) 2.12, s 1.58, s 5.02, q (6.0) 1.43, d (6.0)
12-O-Acyl 2 3 5, 9 6, 8 7
cin 6.76, d (16.0) 7.95, d (16.0) 7.67, d (6.5) 7.43, m 7.40, m
cin 6.88, d (15.5) 8.04, d (15.5) 7.75, d (7.0) 7.41, m 7.38, m
20-O-Acyl 2 3 4 5
tig
ac 2.01, s
2.65, s
7.01, q (7.0) 1.51, d (7.0) 1.79, s d-Cym
1 2 3 4 5 6
5.30, d (9.5) 1.73, m; 2.16, m 3.98, m 3.54, dd (9.5, 2.5) 4.24, m 1.38, d (6.0)
5.29, d (9.5) 1.69, m; 2.16, m 3.95, m 3.47, dd (9.5, 2.5) 4.22, m 1.36, d (6.0)
5.19, d (9.5) 1.76, m; 2.22, m 4.02, m 3.45, dd (9.5, 3.0) 4.23, m 1.40, d (6.0)
5.19, d (9.0) 1.72, m; 2.23, m 4.03, m 3.46, dd (9.5, 2.5) 4.17, dq (9.5, 6.5) 1.37, d (6.0)
OCH3
3.62, s
3.60, s
3.63, s
3.63, s
d-Cym 1 2 3 4 5 6
5.12, d (9.5) 1.78, m; 2.17, m 4.03, m 3.88, dd (10.5, 5.0) 4.22, m 1.58, d (6.5)
5.08, d (10.0) 1.75, m; 2.18, m 4.05, m 3.84, m 4.19, m 1.55, d (6.0)
5.12, d (10.0) 1.78, m; 2.24, m 4.08, m 3.91, m 4.21, m 1.58, d (6.0)
5.13, d (10.0) 1.80, m; 2.26, m 4.08, m 3.61, dd (10.0, 2.0) 4.23, dq (9.5, 6.0) 1.62, d (6.0)
OCH3
3.58, s
3.56, s
3.59, s
3.58, s
d-Allme
d-Thv
1 2 3 4 5 6
5.15, d (10.0) 3.92, t (9.0) 3.72, t (8.0) 3.54, dd (9.5, 2.5) 3.81, m 1.76, d (6.0)
5.12, d (10.0) 3.88, t (9.0) 3.69, t (8.0) 3.49, dd (10.0, 2.5) 3.74, dq (8.5, 6.0) 1.73, d (6.0)
5.16, d (10.5) 3.93, m 3.75, m 3.53, dd (9.5, 2.0) 3.79, dq (8.5, 6.0) 1.79, d (6.0)
4.80, d (7.5) 3.95, m 3.66, m 3.64, m 3.76, dq (8.5, 6.0) 1.61, d (6.0)
OCH3
3.96, s
3.92, s
3.97, s
3.93, s
4.69, d (7.5) 4.05, m 4.22, m 4.20, m 4.01, m 4.35, dd (11.0, 5.0) 4.52, d (10.5)
4.73, d (7.5) 4.07, m 4.28, m 4.22, m 4.09, m 4.40, dd (11.0, 5.5) 4.58, dd (11.0, 2.5)
d-Glc 1 2 3 4 5 6””
4.72, d (7.5) 4.04, m 4.25, m 4.21, m 4.03, m 4.37, dd (11.5, 5.5) 4.55, dd (11.5, 2.5)
cym: cymaropyranosyl; glc: glucopyranosyl; thv: thevetopyranosyl. a
Allme: 6-deoxy-3-O-methyl--allopyranosyl.
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
2.3.7.
Sarcostin (6)
C21 H34 O6 : colorless needles (MeOH). ESI-MS m/z 405.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 38.9 (C-1), 31.2 (C-2), 70.7 (C-3), 42.2 (C-4), 139.4 (C-5), 118.5 (C-6), 34.8 (C-7), 73.6 (C-8), 44.1 (C-9), 36.9 (C-10), 29.4 (C-11), 70.3 (C-12), 58.4 (C-13), 88.4 (C-14), 34.1 (C-15), 33.6 (C-16), 88.9 (C-17), 10.6 (C-18), 18.3 (C19), 72.5 (C-20), 17.8 (C-21). 1 H NMR (C5 D5 N, 500 MHz): ı 3.93 (1H, m, H-3), 5.40 (1H, br s, H-6), 3.97 (1H, m, H-12), 1.96 (3H, s, H-18), 1.39 (3H, s, H-19), 4.40 (1H, q, J = 6.0 Hz, H-20), 1.51 (3H, d, J = 6.0 Hz, H-21).
2.3.8. 12-O-cinnamoyl-20-O-tigloyl(20S)-pregn-6-ene3ˇ,5˛,8ˇ,12ˇ,14ˇ,17ˇ,20-heptanol (7) C35 H46 O9 : colorless needles (MeOH). ESI-MS m/z 633.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 27.3 (C-1), 28.7 (C-2), 66.7 (C-3), 41.3 (C-4), 75.3 (C-5), 136.8 (C-6), 127.1 (C-7), 74.1 (C-8), 36.4 (C-9), 39.8 (C-10), 23.8 (C-11), 75.7 (C-12), 58.0 (C13), 88.3 (C-14), 33.2 (C-15), 34.5 (C-16), 87.8 (C-17), 12.4 (C-18), 21.8 (C-19), 74.6 (C-20), 15.6 (C-21), 166.9 (C-1 ), 120.5 (C-2 ), 143.7 (C-3 ), 135.1 (C-4 ), 128.7 (C-5 ,9 ), 129.3 (C-6 ,8 ), 130.5 (C-7 ), 166.9 (C-1 ), 129.4 (C-2 ), 138.1 (C-3 ), 14.2 (C-4 ), 12.3 (C-5 ). 1 H NMR (C5 D5 N, 500 MHz): ı 4.32 (1H, m, H-3), 5.98 (1H, d, J = 10.0 Hz, H-6), 6.23 (1H, d, J = 10.0 Hz, H-7), 5.39 (1H, dd, J = 10.0, 4.0 Hz, H-12), 2.12 (3H, s, H-18), 1.60 (3H, s, H19), 5.12 (1H, q, J = 6.0 Hz, H-20), 1.49 (3H, d, J = 6.0 Hz, H-21), 6.75 (1H, d, J = 16.0 Hz, H-2 ), 7.92 (1H, d, J = 16.0 Hz, H-3 ), 7.64 (2H, dd, J = 7.0, 1.5 Hz, H-5 , 9 ), 7.39 (2H, dd, J = 7.0, 6.5 Hz, H-6 , 8 ), 7.36 (1H, ddd, J = 7.0, 6.5, 1.5 Hz, H-7 ), 6.96 (1H, q, J = 6.5 Hz, H-3 ), 1.46 (3H, d, J = 6.5 Hz, H-4 ), 1.77 (3H, s, H5 ). 13 C NMR (DMSO-d , 125 MHz): 26.3 (C-1), 27.4 (C-2), 66.0 6 (C-3), 40.1(C-4), 74.3 (C-5), 135.7 (C-6), 126.1 (C-7), 73.0 (C-8), 34.8 (C-9), 38.4 (C-10), 22.6 (C-11), 74.8 (C-12), 57.1 (C-13), 87.2 (C-14), 32.2 (C-15), 33.4 (C-16), 87.1 (C-17), 11.7 (C-18), 21.3 (C-19), 73.6 (C-20), 15.2 (C-21), 165.7 (C-1 ), 119.3 (C-2 ), 143.5 (C-3 ), 134.1 (C-4 ), 128.4 (C-5 ,9 ), 129.0 (C-6 ,8 ), 130.1 (C-7 ), 166.1 (C-1 ), 128.4 (C-2 ), 137.7 (C-3 ), 14.3 (C-4 ), 12.1 (C-5 ). 1 H NMR (DMSO-d , 500 MHz): ı 3.97 (1H, m, H-3), 5.38 (1H, 6 d, J = 10.0 Hz, H-6), 5.65 (1H, d, J = 10.0 Hz, H-7), 4.72 (1H, dd, J = 10.0, 4.5 Hz, H-12), 1.46 (3H, s, H-18), 0.92 (3H, s, H-19), 4.45 (1H, q, J = 6.0 Hz, H-20), 1.09 (3H, d, J = 6.0 Hz, H-21), 6.21 (1H, d, J = 16.0 Hz, H-2 ), 7.42 (1H, d, J = 16.0 Hz, H-3 ), 7.60 (2H, dd, J = 7.5, 1.5 Hz, H-5 ,9 ), 7.39 (2H, m, H-6 ,8 ), 7.38 (1H, m, H-7 ), 6.75 (1H, q, J = 7.0 Hz, H-3 ), 1.61 (3H, d, J = 7.0 Hz, H-4 ), 1.53 (3H, s, H-5 ).
2.3.9. 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene3ˇ,5˛,8ˇ,12ˇ,14ˇ,17ˇ,20-heptanol (8) C32 H42 O9 : colorless needles (MeOH). ESI-MS m/z 593.1 [M + Na]+ . 13 C NMR (C5 D5 N, 125 MHz): 27.5 (C-1), 28.7 (C-2), 66.6 (C-3), 41.3 (C-4), 75.1 (C-5), 136.5 (C-6), 127.3 (C-7), 74.2 (C-8), 36.5 (C-9), 39.7 (C-10), 23.7 (C-11), 75.7 (C-12), 58.1 (C-13), 88.3 (C-14), 33.1 (C-15), 34.1 (C-16), 87.7 (C-17), 12.5 (C-18), 21.7 (C19), 74.6 (C-20), 15.6 (C-21), 166.9 (C-1 ), 120.5 (C-2 ), 143.8 (C-3 ), 135.1 (C-4 ), 128.6 (C-5 ,9 ), 129.1 (C-6 ,8 ), 130.5 (C-7 ), 169.7 (C1 ), 21.5 (C-2 ). 1 H NMR (C5 D5 N, 500 MHz): ı 4.26 (1H, m, H-3), 5.94 (1H, d, J = 10.5 Hz, H-6), 6.22 (1H, d, J = 10.5 Hz, H-7), 5.36 (1H, dd, J = 10.0, 3.5 Hz, H-12), 2.12 (3H, s, H-18), 1.59 (3H, s, H19), 5.06 (1H, q, J = 6.0 Hz, H-20), 1.47 (3H, d, J = 6.0 Hz, H-21), 6.85 (1H, d, J = 16.0 Hz, H-2 ), 7.97 (1H, d, J = 16.0 Hz, H-3 ), 7.70 (2H, d,
687
J = 7.0 Hz, H-5 ,9 ), 7.38 (2H, m, H-6 ,8 ), 7.35 (1H, m, H-7 ), 2.02 (1H, s, H-2 ).
2.4.
Splenocyte proliferation assay
Single cell suspensions were prepared as previously described [9]. Splenocytes were seeded into four wells of a 96-well flatbottom microtiter plate (Nunc) at a cell density of 1 × 107 per ml in 100 l of complete medium, into which 100 l of 1–4 and CsA (0.01–10 g/ml) were added, followed by Con A (final concentration 5 mg l−1 ), LPS (final concentration 10 mg l−1 ), or medium. The plate was incubated at 37 ◦ C under a humidified atmosphere of 5% CO2 . After 44 h, 50 l of MTT solution (2 g l−1 ) was added to each of the wells, which were incubated for 4 h. The microtiter plates were centrifuged (1400 × g, 5 min), and the untransformed MTT was removed carefully by pipetting. Next, 200 l of a Me2 SO working solution (192 l Me2 SO with 8 l HCl 1 mol l−1 ) was added to each well, and the absorbance (A) was evaluated after 15 min in an ELISA reader at 570 nm with a 630 nm reference.
3.
Results and discussion
The EtOH extract of the stems of S. mucronata was extracted with CHCl3 . The CHCl3 soluble portion was subsequently separated using silica, reversed-phase silica gel column chromatography and semi-prep HPLC to yielded four compounds (1–4). These compounds showed positive Libermann–Buchard and Keller–Kiliani reactions, indicating the presence of a steroidal glycoside with a 2-deoxysugar moiety. Each of the isolates was subjected to detailed spectroscopic analysis to establish their chemical structures. Mucronatoside E (1) was isolated as an amorphous powder. Based on the HRESI-MS data, the molecular formula of 1 was determined to be C48 H78 O21 (1013.4923 [M + Na]+ , calcd. 1013.4928). Mild acid hydrolysis of 1 afforded an aglycone (5), cymarose, d-glucose, and an unidentified sugar (on TLC). The aglycone was identified as 12-O-deacetylmetaplexigenin, by comparison of its spectroscopic data to those in the literature [7]. The NMR (1 H NMR, 13 C NMR, DEPT, HMQC, and HMBC) spectral data of compound 1 showed that it contained four anomeric carbon signals at ı 96.4, 100.4, 104.8, and 106.0, correlating with anomeric protons at ı 5.30 (d, J = 9.5 Hz), 5.12 (d, J = 9.5 Hz), 5.15 (d, J = 10.0 Hz), and 4.72 (d, J = 7.5 Hz), respectively, which indicated that there were four sugar units in the compound. The sugar moieties of 1 were identical to those of stemucronatoside C, by comparison of their 1 H NMR and 13 C NMR data and TLC evidence with those in the literature [7]. The sugar sequence of compound 1 was confirmed by the HMBC spectrum, which showed long-range correlations between (i) H-1 of the -d-cymaropyranose (ıH 5.30, 1H, d, J = 9.5 Hz) and C-3 of the aglycone (ıC 78.0); (ii) H-1 of the -d-cymaropyranose (ıH 5.12, 1H, d, J = 9.5 Hz) and C-4 of the -d-cymaropyranose (ıC 83.1); (iii) H-1 of the 6-deoxy-3-O-methyl--d-allopyranose (abbreviated as allme) (ıH 5.15, 1H, d, J = 10.0 Hz) and C-4 of the -d-cymaropyranose (ıC 83.0); and (iv) H-1 of the ˇ-d-glucopyranose (ıH 4.72, 1H, d, J = 7.5 Hz) and C-4 of the 6-deoxy-3-O-methyl--dallopyranose (ıC 83.4). Thus, the sequence and linkage sites
688
s t e r o i d s 7 1 ( 2 0 0 6 ) 683–690
of the sugar units were established as glc-(1 → 4)-allme(1 → 4)-cym-(1 → 4)-cym-(1 → 3)-aglycone. This assignment was consistent with the ESI-MS data, which showed the quasi-molecular ion at m/z 1013.4 [M + Na]+ , and other prominent fragments at m/z 851.4 [M + Na − glc]+ , 691.3 [M + Na − glc-allme]+ , 547.2 [M + Na − glc-allme-cym]+ , and 403.1, which corresponded to the aglycone moiety. In the 13 C NMR spectrum of 1, glycosidation shifts were observed at C-2, C-3, and C-4 by −2.5, +6.6 and −3.9, respectively, when compared with 13 C chemical shifts of 5, indicating that the sugar chain was linked to the C-3 hydroxyl group of the aglycone. Thus, the structure of 1 was established as 12-O-deacetylmetaplexigenin 3-O--d-glucopyranosyl(1 → 4)-6-deoxy-3-O-methyl--d-allopyranosyl-(1 → 4)--dcymaropyranosyl-(1 → 4)--d-cymaropyranoside. Mucronatoside F (2) possessed the molecular formula C48 H80 O21 (1015.5103 [M + Na]+ , calcd. 1015.5084) on the basis of the HRESI-MS. Mild acid hydrolysis of 2 afforded an aglycone (6) and a sugar mixture. 6 was identical to sarcostin by comparison of its spectroscopic data with those in the literature [10]. In 1975, the absolute configuration of 6 was confirmed by the X-ray analysis of its 3-O-p-bromobenzoate which showed S-configuration at C-20 by Hayashi and Mitsuhashi et al. [11]. Moreover, in the 20-hydroxy-C/d-cis-pregnane type steroids the free rotation of the side-chain is considered to be restricted by steric hindrance among the C-18 methyl, C-21 methyl and 20-hydroxyl groups on the basis of a CPK model [12]. In compound 6, the C-17 OH further restricted the rotate of the side-chain. Thus, the NOESY evidence of (i) H-12 at ı 3.97 and H-20 at ı 4.40; (ii) H-16␣ at ı 1.90 and H-20 at ı 4.40; and (iii) H-16 at ı 1.85 and H-21 at ı 1.51 can further confirm the 20-S configuration. The 1 H and 13 C NMR spectral data for the aglycone moiety of 2 were similar to those of sarcostin (6) with the major difference being the absence of signals of an OH group at C-3. The only other difference in the 13 C NMR data between 2 and 6 occurred for C-2, C-3, and C-4, by −2.0, +7.2, and −2.7, respectively. Therefore, the sugar moiety was linked to the C-3 hydroxyl group of the aglycone. The NMR assignments of 2 were made unambiguously on the basis of HMQC, HMBC and 1 H, and 1 H COSY experiments; by comparison to those of 1, 2 was determined to have the same sugar sequence in the oligosaccharide moiety. Thus, compound 2 was established as sarcostin 3-O--d-glucopyranosyl(1 → 4)-6-deoxy-3-O-methyl--d-allopyranosyl-(1 → 4)--dcymaropyranosyl-(1 → 4)--d-cymaropyranoside. Mucronatoside G (3) possessed the molecular formula C62 H92 O24 (1243.5894 [M + Na]+ , calcd. 1243.5871) on the basis of the HRESI-MS. Mild acid hydrolysis of 3 afforded an aglycone (7) and a sugar mixture. The molecular formula of 7 was deduced as C35 H46 O9 by the quasi-molecular ion peak at m/z 633.1 [M + Na]+ in the ESI-MS spectra and by its 13 C NMR data. 7 was identical to 12-O-cinnamoyl-20-O-tigloyl(20S)pregn-6-ene-3,5␣,8,12,14,17,20-heptanol by comparison of its spectroscopic data with those in the literature [13], but the C-1 chemical shifts of the cinnamoyl group and the tigloyl group were both at 166.9 ppm. In order to locate the cinnamoyl group and tigloyl group in 7, a HMBC experiment (in DMSO-d6 ) had to be carried out. In the HMBC spectrum, the following long-range correlations were observed between (i) H-12 (ıH 4.72) and C-1 of the cinnamoyl (ıC 165.7); and (ii)
H-20 (ıH 4.45) and C-1 of the tigloyl (ıC 166.1). Thus, 7 was determined to be 12-O-cinnamoyl-20-O-tigloyl (20S)-pregn-6ene-3,5␣,8,12,14,17,20-heptanol. The 1 H and 13 C NMR spectral data for the aglycone moiety of compound 3 were similar to those of 7 with the major difference being the absence of signals for an OH group at C-3. The only other difference in 13 C NMR spectral data between 3 and 7 occurred for C-atoms C-2, C-3, and C-4, by −2.1, +8.2, and −2.2, respectively. The 1 H and 13 C NMR signals due to the sugar moieties of 3 were also superimposable onto those of 1. Consequently, the structure of 3 was established as 12-O-cinnamoyl-20-Otigloyl(20S)-pregn-6-ene-3,5␣,8,12,14,17,20-heptanol 3O--d-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl--d-allopyranosyl-(1 → 4)--d-cymaropyranosyl-(1 → 4)--dcymaropyranoside. Mucronatoside H (4) had the molecular formula C53 H78 O19 (1041.4990 [M + Na]+ , calcd. 1041.5030) on the basis of the HRESI-MS data. Mild acid hydrolysis of 4 afforded an aglycone (8), cymarose, and an unidentified sugar (on TLC). The molecular formula of 8 was deduced as C32 H42 O9 by the quasimolecular ion peak at m/z 593.1 [M + Na]+ in the ESI-MS spectra and its 13 C NMR data. The spectral analysis of 8 revealed a distinct similarity to 7. In comparison to 7, 8 showed the absence of the tigloyl group, but suggested the presence of an acetyl group. In the HMBC spectrum of 8, the following long-range correlations were observed: (i) between H-12 (ıH 5.36) and C-1 of the cinnamoyl (ıC 166.9), and (ii) between H-20 (ıH 5.06) and C-1 of the acetyl (ıC 169.7). Thus, 8 was determined to be 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6ene -3,5␣,8,12,14,17,20-heptanol. The NMR (1 H NMR, 13 C NMR, DEPT, HMQC, and HMBC) spectral data of compound 4 showed that it contained three anomeric carbon signals at ı 97.7, 100.3, and 106.2, correlating with anomeric protons at ı 5.19 (d, J = 9.0 Hz), 5.13 (d, J = 10.0 Hz), and 4.80 (d, J = 7.5 Hz), respectively, which indicated that there were three sugar units in compound 1. The sugar moieties of 4 were identical to those of stemucronatoside B, deduced by comparing of their 1 H and 13 C NMR data and TLC data to those in the literature [7]. The sequence of sugars in 4 was confirmed by the HMBC spectrum, which showed distinct cross-peaks of correlation between (i) H-1 of the -d-cymaropyranose (ıH 5.19, 1H, d, J = 9.0 Hz) and C-3 (ıC 74.9); (ii) H-1 of the -d-cymaropyranose (ıH 5.13, 1H, d, J = 10.0 Hz) and C-4 of the -d-cymaropyranose (ıC 82.9); and (iii) H-1 of the -d-thevetopyranose (ıH 4.80, 1H, d, J = 7.5 Hz) and C-4 of the -d-cymaropyranose (ıC 83.0). The glycosidation shifts of the carbon signals of 4 were observed at C-2, C-3, and C-4, by −2.1, +8.3, and −2.3, respectively, compared to 8, indicating the sugar chain linked to the C-3 hydroxyl group of the aglycone. Consequently, the structure of 4 was established as 12-O-cinnamoyl-20-O-acetyl(20S)-pregn-6-ene3,5␣,8, 12,14,17,20-heptanol 3-O--d-thevetopyranosyl(1 → 4)--d-cymaropyranosyl-(1 → 4)--d-cymaropyranoside. Comparing the 13 C-NMR data of compounds 1–4 (Table 1), the chemical shifts of the C-1 and C-9 of compounds 3 and 4 are ca. 12 and 9 ppm high-field shifted than that of compounds 1 and 2, respectively. Those phenomena could be relate to that (i) compounds 3 and 4 had the 5␣-OH group, which make the ␥-position carbons C-1 and C-9 up-field, and (ii) C-1 and C-9 in compounds 3 and 4 were in the shielding regions of the double bond between C-6 and C-7.
steroids
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Fig. 2 – Effect of compounds 1–4 on Con A-stimulated splenocyte proliferation in vitro. Splenocytes were cultured with the various concentrations of compounds 1–4 or CsA and Con A (5 g/ml) in RPMI 1640 medium for 48 h. Cellular proliferation was measured by the MTT method as described in the text and shown as a stimulation index. The values are presented as means ± S.E. (n = 4). Significant differences compared to 0 g/ml are designated as a P < 0.05, b P < 0.01, and c P < 0.001. CsA: cyclosporin A (positive drug).
Fig. 3 – Effect of compounds 1–4 on LPS-stimulated splenocyte proliferation in vitro. Splenocytes were cultured with the various concentrations of compounds 1–4 or CsA and LPS (10 g/ml) in RPMI 1640 medium for 48 h. Cellular proliferation was measured by the MTT method as described in the text and shown as a stimulation index. The values are presented as means ± S.E. (n = 4). Significant differences compared to 0 g/ml are designated as a P < 0.05, b P < 0.01, and c P < 0.001. CsA: cyclosporin A (positive drug).
The effects of 1–4 on mitogen-stimulated mice splenocyte proliferation in vitro are shown in Figs. 2 and 3. Compounds 2 and 4 significantly inhibited Con A- and LPS-stimulated mice splenocyte proliferation in a dose-dependent manner. However, compound 1 and 3 significantly enhanced Con A- and LPS-induced mice splenocyte proliferation at suitable concentrations. There were no significant differences in inhibitory effect on mitogen-stimulated splenocyte proliferation among CsA, compounds 2 and 4 at the concentration of 1 g/ml. CsA showed cytotoxic activity mice splenocyte at the concentration of 10 g/ml, while compounds 2 and 4 had no influences on mice splenocyte growth at the same concentration. The inhibitory effect compounds 2 and 4 on mitogen-stimulated splenocyte proliferation at the concentration of 10 g/ml was more significant than that of CsA at the concentration of 1 g/ml (P < 0.05).
Acknowledgements We gratefully acknowledge the Zhejiang Provincial Natural Science Foundation of China (Grant-in Aid No. M303749) and the Zhejiang Provincial Science and Technology Council (Grant-in Aid No. 2004F13G1360002) for financial support.
references
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