Planta Med 2017; 83(01/02): 126-134
DOI: 10.1055/s-0042-108057
Natural Product Chemistry and Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Polyoxypregnane Glycosides from the Roots of Marsdenia tenacissima and Their Anti-HIV Activities

Xu Pang*
1  Beijing Institute of Radiation Medicine, Beijing, China
2  Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
,
Li-Ping Kang*
1  Beijing Institute of Radiation Medicine, Beijing, China
3  Tianjin University of Traditional Chinese Medicine, Tianjin, China
,
Xiao Mei Fang*
2  Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
,
Yang Zhao
1  Beijing Institute of Radiation Medicine, Beijing, China
,
He-Shui Yu
3  Tianjin University of Traditional Chinese Medicine, Tianjin, China
,
Li-Feng Han
3  Tianjin University of Traditional Chinese Medicine, Tianjin, China
,
Hai-Tao Li
4  Yunnan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Jinghong, China
,
Li-Xia Zhang
4  Yunnan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Jinghong, China
,
Bao-Lin Guo
5  Institute of Medicinal Plant development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
,
Li-Yan Yu
2  Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
,
Bai-Ping Ma
1  Beijing Institute of Radiation Medicine, Beijing, China
› Author Affiliations
Further Information

Correspondence

Prof. Baiping Ma
Beijing Institute of Radiation Medicine
No. 27 Tai-ping Road, Haidian District
Beijing 100850
Peopleʼs Republic of China
Phone: +86 10 68 21 00 77   
Fax: +86 10 68 21 46 53   

 

Prof. Lixia Zhang
Yunnan Branch of Institute of Medicinal Plant Development
Chinese Academy of Medical Sciences & Peking Union Medical College
NO. 138 Xuanwei Street
Jinghong 666100
China

Publication History

received 14 July 2015
revised 17 April 2016

accepted 19 April 2016

Publication Date:
07 June 2016 (online)

 

Abstract

A continuous phytochemical study on the roots of Marsdenia tenacissima led to the isolation and identification of 13 new polyoxypregnane glycosides named marstenacissides B10–B17 (1, 2, 4, 7, 8, 11, 12, and 14) and marstenacissides A8-A12 (3, 9, 10, 13, and 15) in addition to two known polyoxypregnane glycosides marsdenosides M and L (5 and 6). Their structures were established by spectroscopic techniques and by comparison with the reported data in the literature. Moreover, the anti-HIV activities of these isolates and the previous isolated marstenacissides A1–A7 and B1–B9 were assessed, some of which exhibited slight or negligible effects against HIV-1.


#

Introduction

Marsdenia tenacissima (Roxb.) Moon (Asclepiadaceae), a perennial climber recorded in the Flora of China, is extensively distributed in the Yunnan province of China. The roots of this plant are widely used as a traditional herbal medicine of Dai nationality, called “Dai-Bai-Jie” in Chinese, due to its pharmacological functions of clearing heat, expelling miasma, decreasing swelling, alleviating pain, etc. In addition, these roots are also used as the main medicinal materials for preparing a series of preparations [1]. The isolation and structural identification of 16 polyoxypregnanes glycosides (marstenacissides A1–A7 and B1–B9) from the roots of M. tenacissima have been reported in our previous paper [2]. A continuous phytochemical study on the roots of M. tenacissima led to the further isolation of 13 new chemicals (14 and 715) and 2 known polyoxypregnane glycosides (5 and 6; [Fig. 1]).

Zoom Image
Fig. 1 Structures of 115.

The traditional Chinsese medicine with functions of heat clearing and detoxicating is supposed to have specific antiviral activity based on the correlation analysis between this kind of traditional medicine and antiviral drugs [3]. Therefore, the extracts of M. auricularis roots were subjected to a preliminary screening of activity against HIV-1, and it was found that the 95 % EtOH extract of M. auricularis roots, especially its EtOAc-soluble fraction, exhibited a significant HIV-1 inhibitory effect. In view of the above significant HIV-1 inhibitory effect, all 31 isolated polyoxypregnane glycosides were screened, followed by chemical work.


#

Results and Discussion

After a series of purification steps, 15 compounds were obtained from the roots of M. auricularis, and all of the compounds were identified to be C21 steroidal glycosides with 2-deoxysugar units. Among them, two known compounds were marsdenosides M (5) and L (6), determined by comparing their 13C NMR data with the literature reported [4], and the other compounds (14 and 715) were identified to be new polyoxypregnane glycosides by analysis of NMR (including 1D and 2D NMR) and MS spectra.

Compound 1 had a molecular formula of C59H84O24 based on the HRESIMS ion [M + Na]+ at m/z 1199.5303. In the 1H NMR spectrum, three methyl singlet signals at δ 1.55 (3 H, s, H-18), 1.54 (3 H, s, H-19), and 1.99 (3 H, s, H-21) and three signals at δ 3.80 (1 H, m, H-3), 5.97 (1 H, t, J = 10.1 Hz, H-11), and 5.59 (1 H, d, J = 10.1 Hz, H-12) corresponding to carbons with secondary oxidation were observed. Combination of 1H and 13C NMR data indicated 1 had a C21 steroidal skeleton. The proton signals at δ 6.77 (1 H, qq, J = 7.1, 1.4 Hz, Tig-H-3), 1.33 (3 H, d, J = 7.1, Tig-H-4), 1.50 (3 H, s, Tig-H-5), 8.15 (2 H, dd, J = 7.8, 1.3 Hz, Bz-H-3, 7), 7.36 (2 H, dd, J = 7.8, 7.4 Hz, Bz-H-4, 6), and 7.46 (1 H, t, J = 7.4 Hz, Bz-H-5) as well as two groups of characteristic carbon signals at δ 167.0 (Tig-C-1), 128.6 (Tig-C-2), 138.7 (Tig-C-3), 14.1 (Tig-C-4), and 11.7 (Tig-C-5), 166.5 (Bz-C-1), 130.6 (Bz-C-2), 130.0 (Bz-C-3, 7), 128.8 (Bz-C-4, 6), and 133.5 (Bz-C-5) indicated the existence of a tigloyl and a benzyl group in the molecular structure. By the combined use of HSQC, HMBC, and 1H-1H COSY experiments ([Fig. 2]), the proton and carbon signals of the aglycone moiety were assigned. In the ROESY spectrum, the dipolar interaction of H-11/H-19, H-12/H-17, H-12/H-9, H-21/H-18, H-9/H-4a, H-9/H-1a, H-3/H-4a, H-3/H-1a, and H-3/H-2a deduced the orientations of H-3, H-11, H-12, and H-17 to be α, β, α, and α, respectively ([Fig. 3]). Therefore, the aglycone structure of 1 was identified as 8β,14β-epoxy-3β,11α,12β-trihydroxyprengn-5-en-20-one, which is a new C21 steroid skeleton. On the basis of HMBC correlations between δ 167.0 (Tig-C-1) and 5.97 (H-11), and between δ 166.5 (Bz-C-1) and 5.59 (H-12), the position of the tigloyl and benzyl groups were determined at C-11 and C-12, respectively ([Fig. 2]). The anomeric regions in the 1H and 13C NMR spectra presented four protons at δ 5.24 (1 H, d, J = 8.8 Hz), 5.19 (1 H, d, J = 7.9 Hz), 4.90 (1 H, d, J = 7.8 Hz), and 4.76 (1 H, dd, J = 9.5, 1.4 Hz) and four carbon signals at δ 106.6, 105.0, 101.9, and 97.9, suggesting the existence of four sugar units in 1. The β configurations of the four sugars were determined based on each large coupling constant (3 J 1, 2 > 7 Hz). Moreover, two methyl doublets at δ 1.56 (3 H, d, J = 5.3 Hz), 1.61 (3 H, d, J = 6.2 Hz) and two methyl singlets at δ 3.48 (3 H, s), 3.79 (3 H, s) in the 1H NMR spectrum indicated that two of the four sugar units were 6-deoxy-3-O-methly pyranoses [5]. Eventually, the sugar units were identified as oleandrose, 6-deoxy-3-O-methyl-allose, and two glucoses by NMR spectroscopic data analysis as well as by comparison with previously reported values. Using 1H-1H COSY, HSQC, and HMBC spectra, the proton and carbon resonances of each sugar were fully assigned. The connectivity of the sugars was established by HMBC correlations between δ 5.24 (Allo-H-1) and 83.3 (Ole-C-4), between δ 4.90 (Glc1-H-1) and 83.4 (Allo-C-4), and between δ 5.19 (Glc2-H-1) and 81.5 (Glc1-C-4) ([Fig. 2]). Therefore, the sugar moiety could be deduced as 3-O-β-glucopyranosyl-(1 → 4)-β-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1 → 4)-β-oleandropyranoside, well in agreement with those in the compounds isolated from the same plant [6], [7], [8]. Also, previous phytochemical studies suggested that the absolute configuration of the glucose, 6-deoxy-3-O-methyl-allose, and oleandrose from M. auricularis should be D [4], [5], [6], [7], [8], [9], [10], [11]. Furthermore, the glycosidation site was determined by HMBC correlations between δ 4.76 (Ole-H-1) and 77.0 (C-3) ([Fig. 2]). Consequently, the structure of 1 was elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-tigloyl-12-O-benzyl-8β,14β-epoxy-3β,11α,12β-trihydroxyprengn-5-e-20-one and named marstenacisside B10.

Zoom Image
Fig. 2 Key HMBC and 1H-1H COSY correlations for 1.
Zoom Image
Fig. 3 Key ROESY correlations of the aglycone for 1.

Compound 2, with the molecular formula of C57H86O24 determined by the HRESIMS ion [M + Na]+ at m/z 1177.5454, had the same sugar moiety and C21 steroid skeleton as 1 based on comparison of their NMR data. In the 13C NMR spectrum of 2, the characteristic carbon signals at δ 167.1, 167.0, 138.6, 138.4, 128.9, 128.8, 14.3, 14.2, 12.0, and 12.0 suggested the existence of double tigloyl groups in 2. By the combined use of HSQC, HMBC, and 1H-1H COSY experiments, the structure of 2 was finally elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11,12-O-ditigloyl-8β,14β-epoxy-3β,11α,12β-tridroxypregnane-5-en-20-one and named marstenacisside B11.

Compound 3, with the molecular formula of C53H74O19 determined by the HRESIMS ion [M – H]at m/z 1013.4787, had the same C21 steroid skeleton and ester groups as 1 by comparing their NMR data. The 1H and 13C NMR spectra of 3 showed three anomeric proton signals at δ 4.78 (1 H, d, J = 9.4 Hz), 4.99 (1 H, d, J = 7.7 Hz), and 5.27 (1 H, d, J = 8.0 Hz) and three anomeric carbon signals at δ 97.9, 101.9, and 106.6, suggesting that the sugar moiety of 3 was composed of three sugar units. The large coupling constants (3 J 1, 2 > 7 Hz) deduced the β configurations of the three sugars. Finally, the sugar units were identified as oleandrose, 6-deoxy-3-O-methyl-allose, and glucose by NMR spectroscopic data analysis as well as by comparison with previously reported values. Using 1H-1H COSY, HSQC, and HMBC spectra, the proton spin systems and the carbon resonances of three sugars were fully assigned. The HMBC correlations between δ 5.27 (Allo-H-1) and 83.2 (Ole-C-4), and between δ 4.99 (Glc-H-1) and 83.3 (Allo-C-4) established the sequence of the sugar chain. Therefore, the sugar moiety could be deduced as 3-O-β-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-allopyranosyl-(1 → 4)-β-oleandropyranoside and well coincided with neo-condurangotriose in other compounds that were also isolated from M. tenacissima [9]. The glycosidation site was confirmed by the HMBC correlation between δ 4.78 (Ole-H-1) and 76.2 (C-3). Finally, the structure of 3 was elucidated to be 3-O-β-D-glucopyranosyl-(1 → 4)- β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-tigloyl-12-O-benozyl-8β,14β-epoxy-3β,11α,12β-tridroxypregnane-5-en-20-one and named marstenacisside A8.

Compound 4 had the molecular formula C59H86O24, determined by the HRESIMS ion [M – H] at m/z 1177.5488, with two mass units more than that of 1. NMR data comparison suggested that 4 and 1 had an almost identical structure except for the difference in the A-ring portion. In the 13C NMR spectrum of 4, the carbon signals of C-3 (δ 76.0), C-4 (δ 35.0), C-5 (δ 44.2), C-6 (δ 27.4), C-7 (δ 32.5), and C-19 (δ 13.1) deduced that 4 had the sp 3 carbons of C-5 and C-6. Thus, the C21 steroid skeleton of 4 could be deduced as 5α-8β,14β-epoxy-3β,11α,12β-tridroxypregnane-20-one, which is well in agreement with 17β-tenacigenin B [5], [11]. Confirmed by the combined use of 1H-1H COSY, HSQC, and HMBC experiments, the structure of 4 was finally elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-tigloyl-12-O-benozyl-5α-8β,14β-epoxy-3β,11α,12β-tridroxypregnane-20-one and named marstenacisside B12.

Compound 7, with a molecular formula of C59H88O25 determined by the HRESIMS ion [M – H] at m/z 1195.5596, had two hydrogen atoms and one oxygen atom more than 4. The 13C NMR data of 7 suggested that it had almost the same carbon signals as 4 except for significant differences at C-8 and C-14. The difference of molecular formula and chemical shifts at C-8 (δ 78.4, + 12.1 ppm) and C-14 (85.6, + 13.9 ppm) deduced, in the structure of 7, two free hydroxyl groups linked to the C-8 and C-14 positions. Therefore, the C21 steroid skeleton of 7 could be deduced as 5α-3β,11α,8β,12β,14β-pentadroxypregnane-20-one, which well coincided with tenacigenin C [10], [11], [12]. Confirmed by the combined use of 1H-1H COSY, HSQC, and HMBC experiments, the structure of 7 was elucidated to be 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-tigloyl-12-O-benozyl-5α-3β,11α,8β,12β,14β-pentadroxypregnane-20-one and named marstenacisside B13.

Compound 8 had the same molecular formula of C59H88O25 as 7, which was determined by the HRESIMS ion [M – H] at m/z 1195.5588. The NMR data of 8 and 7 revealed that they had almost identical carbon signals. However, the carbon signals of 8 at δ 72.2 (C-11) and 78.8 (C-12) obviously differed from those of 7 at δ 71.3 (C-11) and 79.8 (C-12), suggesting they had the same ester groups but with different esterification positions. The HMBC correlations between δ 6.67 (H-11) and 166.0 (Bz-C-1), and between δ 5.55 (H-12) and 170.0 (Tig-C-1) determined the linkages of the benzoyl group to C-11 and the tigloyl group to C-12. By analyses of 1H-1H COSY, HSQC, and HMBC spectra, the NMR data of 8 were fully assigned. Thus, the structure of 8 was determined as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-benozyl-12-O-tigloyl-5α-3β,11α,8β,12β,14β-pentadroxypregnane-20-one and named marstenacisside B14.

Compound 9, with the molecular formula of C53H78O20 determined by the HRESIMS ion [M – H] at m/z 1033.5083, had the same aglycone moiety as 7 by comparison of their NMR data. In the 1H and 13C NMR spectra of 9, three anomeric protons at δ 4.82 (1 H, d, J = 9.7 Hz), 4.99 (1 H, d, J = 7.7 Hz), and 5.29 (1 H, d, J = 8.0 Hz) and three anomeric carbons at δ 97.5, 101.9, and 106.6 were observed. Compound 9 had the same sugar moiety as 3 by comparing their NMR data of the sugar moiety. Therefore, the structure of 9 was elucidated to be 3-O-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-tigloyl-12-O-benozyl-5α-3β,11α,8β,12β,14β-pentadroxypregnane-20-one and named marstenacisside A9.

Compound 10, with the molecular formula of C53H78O20 determined by the HRESIMS ion [M – H] at m/z 1033.5059, was an isomer of 9. The NMR data suggested that 10 had almost identical carbon signals as 9. The carbon signals of 10 at δ 72.2 (C-11) and 78.8 (C-12), obviously different with those of 9 at δ 71.3 (C-11) and 79.8 (C-12), suggested that they had the same ester groups but with different esterification positions. Further comparison of the aglycone NMR data of 10 and 8 deduced that they had the same aglycone. Consequently, the structure of 10 was elucidated to be 3-O-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-benozyl-12-O-tigloyl-5α-3β,11α,8β,12β,14β-pentadroxypregnane-20-one and named marstenacisside A10.

Compound 11 had a molecular formula of C56H82O24 as determined by the HRESIMS ion [M – H] at m/z 1137.5137. The NMR data of 11 suggested that it had the same sugar moiety as 1. In the 13C NMR spectra of 11, two olefinic carbon signals at δ 139.7 and 122.6 deduced the double bond at C-5 and C-6, and four oxidative carbons at δ 77.3 (C-3), 71.8 (C-11), 78.6 (C-12), and 84.1 (C-14) suggested the existence of four hydroxyl groups in the aglycone. By comparing the NMR data of 11 with those compounds in the literature, the C21 steroid skeleton of 11 was deduced to be 3β,11α,12β,14β-tetrahydroxyprengn-5-en-20-one, well in agreement with drevogenin P [13]. Characteristic carbon signals at δ 170.2 and 21.3 along with 166.8, 133.4, 130.2 (× 2), 130.1, and 129.2 (× 2) indicated the existence of an acetyl group and a benzyl group in the molecular structure. Based on the HMBC correlations between δ 170.4 (Ac-C-11) and 5.85 (H-11), and between δ 166.8 (Bz-C-1) and 5.43 (H-12), the linkages of an acetyl group to C-11 and a benzoyl group to C-12 were determined. Finally, confirmed by the combined use of 1H-1H COSY, HSQC, and HMBC experiments, the structure of 11 was elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-acetyl-12-O-benozyl-3β,11α,12β,14β-tetrahydroxyprengn-5-en-20-one and named marstenacisside B15.

Compound 12, with a molecular formula of C57H88O24 as determined by the HRESIMS ion [M – H] at m/z 1155.5636, had the same C21 steroidal skeleton structure as 11 by comparing their NMR data. In the 13C NMR spectrum of 12, characteristic carbon signals at δ 167.9, 167.1, 138.6, 138.5, 129.0, 128.5, 14.3, 14.3, 12.1, and 12.0 suggested the existence of double tigloyl groups in the molecule. By the combined use of 1H-1H COSY, HSQC, and HMBC experiments, the structure of 12 was elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11,12-O-ditigloyl-3β,11α,12β,14β-tetrahydroxyprengn-5-en-20-one and named marstenacisside B16.

Compound 13 had a molecular formula of C53H76O19 determined by the HRESIMS ion [M – H] at m/z 1015.4949. The NMR data of 13 suggested that it had same C21 steroid skeleton as 11 and 12. In the 13C NMR spectrum of 13, two groups of characteristic carbon signals at δ 166.2, 133.4, 130.8, 130.1 (× 2), and 128.8 (× 2) and at δ 167.8, 128.3, 138.8, 14.2, and 11.9, coincided well with those of 8, suggesting that 13 had a benzoyl group and a tigloyl group in the molecule. By comparing the carbon chemical shifts of the ester groups as well as the carbon chemical shifts of C-11 and C-12, the linkages of benzoyl to C-11 and tigloyl to C-12 were also the same as those in 8. Moreover, the sugar moiety of 13 was deduced to be the same as that of 3 by comparing their NMR data. Thus, the structure of 13 was elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-benozyl-12-O-tigloyl-3β,11α,12β,14β-tetrahydroxyprengn-5-en-20-one and named marstenacisside A11.

Compound 14 had a molecular formula of C56H84O24 as determined by the HRESIMS ion [M – H] at m/z 1139.5333, two mass units more than that of 11. The NMR data of 14 suggested that it had the almost identical C21 steroid skeleton as 11, except for the significant difference in the A-ring portion. In the 13C NMR spectrum of 14, carbon signals of C-3 (δ 76.2), C-4 (δ 35.5), C-5 (δ 44.7), C-6 (δ 29.4), C-7 (δ 28.4), and C-19 (δ 12.4) indicated that 14 had the sp 3 carbons of C-5 and C-6. Thus, the C21 steroid skeleton was deduced to be 5α-3β,11α,12β,14β-tetrdroxypregnane-20-one, well in agreement with decaylconduragogenin A [12]. Therefore, by using 1H-1H COSY, HSQC, and HMBC experiments, the structure of 14 was elucidated as 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-acetyl-12-O-benozyl-5α-3β,11α,12β,14β-tetrdroxypregnane-20-one and named marstenacisside B17.

Compound 15 had a molecular formula of C53H78O19 as determined by the HRESIMS ion [M – H] at m/z 1017.5085. The NMR data suggested that its C21 steroid skeleton is same as that of 14. In the 13C NMR spectrum of 15, two groups of characteristic carbon signals at δ 167.4, 128.9, 138.6, 14.1, and 11.7 and at δ 166.8, 133.7, 130.3, 130.1 (× 2), and 128.9 (× 2) suggested that it had a tigloyl group and a benzoyl group. The linkages of the tigloyl group to C11 and the benzoyl group to C-12 were determined by the HMBC correlations between δ 167.4 (Tig-C-1) and 5.87 (H-11), and between 166.8 (Bz-C-1) and 5.43 (H-12). The sugar moiety of 15 was deduced to be the same as 3 by comparing the NMR data. Consequently, confirmed by the combined use of 1H-1H COSY, HSQC, and HMBC experiments, the structure of 15 was elucidated as 3-O-β-D-glucopyranosyl-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-11-O-tigloyl-12-O-benozyl-5α-3β,11α,12β,14β-tetrdroxypregnane-20-one and named marstenacisside A12.

Isolates 115 above and the previously isolated 16 polyoxypregnane glycosides, marstenacissides A1–A7 and B1–B9 [2], were further evaluated for anti-HIV and cytotoxic activities. As illustrated in [Table 1], treatment of HIV-1-infected SupT1 cells with 6, 11, 12, 14, marstenacissides A3, A4, and marstenacissides B1, B3, and B9 (30 µM) slightly inhibited HIV-1 replication, indicating that these compounds only had a marginal inhibitory effect on HIV-1, while the other compounds exhibited negligible effects since their inhibition rates were far below average. The toxicity test showed that all 31 compounds at the concentration of 30 µM displayed different cytoxicities against SupT1 cells (cell survival rate from 30 % to 71 %), while no linear relationship between toxicity and anti-HIV activity was observed.

Table 1 HIV-1 inhibition rate and cell survival rate (x̄ ± s, n = 3) of 31 polyoxypregnane glycosides from the roots of M. tenacissima.

Compounds

Dose

Inhibition rate (%)

Cell survival rate (%)

Efavirenz

0.01 µM

99.91 ± 0.10

99.51 ± 0.03

1

30 µM

13.70 ± 4.66

43.37 ± 14.98

2

30 µM

20.90 ± 5.17

50.21 ± 5.94

3

30 µM

13.478 ± 4.66

53.18 ± 13.03

4

30 µM

21.19 ± 5.36

49.36 ± 2.12

5

30 µM

8.26 ± 2.77

57.47 ± 5.78

6

30 µM

39.26 ± 5.56

48.56 ± 13.77

7

30 µM

29.09 ± 1.87

42.48 ± 1.03

8

30 µM

31.36 ± 6.96

57.86 ± 3.49

9

30 µM

29.77 ± 5.90

71.21 ± 8.46

10

30 µM

25.57 ± 0.99

35.62 ± 9.40

11

30 µM

47.75 ± 4.73

39.26 ± 0.54

12

30 µM

37.81 ± 4.23

58.56 ± 3.15

13

30 µM

20.06 ± 2.46

38.64 ± 5.81

14

30 µM

36.87 ± 8.27

41.10 ± 10.73

15

30 µM

25.52 ± 3.87

64.60 ± 8.02

Marstenacisside A1

30 µM

14.12 ± 1.75

42.05 ± 6.51

Marstenacisside A2

30 µM

16.48 ± 1.39

34.07 ± 1.68

Marstenacisside A3

30 µM

53.48 ± 9.48

30.29 ± 1.91

Marstenacisside A4

30 µM

39.74 ± 4.56

50.61 ± 0.88

Marstenacisside A5

30 µM

24.78 ± 4.41

38.12 ± 3.01

Marstenacisside A6

30 µM

25.14 ± 7.21

29.44 ± 6.52

Marstenacisside A7

30 µM

15.63 ± 3.82

69.90 ± 4.80

Marstenacisside B1

30 µM

41.32 ± 5.32

51.75 ± 3.61

Marstenacisside B2

30 µM

28.35 ± 3.69

56.53 ± 11.76

Marstenacisside B3

30 µM

48.83 ± 7.96

45.64 ± 1.69

Marstenacisside B4

30 µM

17.02 ± 6.04

62.22 ± 5.64

Marstenacisside B5

30 µM

33.81 ± 8.56

31.55 ± 2.68

Marstenacisside B6

30 µM

24.15 ± 5.03

39.63 ± 8.07

Marstenacisside B7

30 µM

22.54 ± 6.11

55.77 ± 2.13

Marstenacisside B8

30 µM

32.42 ± 6.73

60.34 ± 3.64

Marstenacisside B9

30 µM

39.20 ± 5.73

48.32 ± 0.61

The EtOAc-soluble fraction of the 95 % EtOH extract from M. auricularis roots showed a significant HIV-1 inhibitory effect, while all of the isolated polyoxypregnane glycosides showed marginal and negligible inhibitory effects on HIV-1, which suggested that a further chemical investigation on this traditional medicine is needed to explore bioactive constituents against HIV-1.


#

Materials and Methods

General experimental procedures

NMR spectra were recorded on a Bruker DRX-500 spectrometer (500 MHz for 1H NMR and 125 MHz for 13C-NMR) and a Varian UNITY INOVA 600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C-NMR) in pyridine-d 5 (Sigma-Aldrich), and the chemical shifts are given in δ (ppm). HRESIMS was performed on the Synapt MS (Waters Corporation). Optical rotations were measured with the Perkin-Elmer 343 polarimeter (PerkinElmer) and JASCO J-810 polarimeter (JASCO Corporation). UV data were recorded on a UV-2500 spectrophotometer (MAPADA Corporation). HPLC analyses were performed on an Agilent 1100 series (Agilent Technologies) equipped with an Alltech 2000 evaporative light scattering detector (temp: 110 °C, gas: 2.4 L/min, Alltech Corporation) and a Techmate C18 column (4.6 mm × 250 mm, ODS, 5 µm, Techmate Co. Ltd.). Semipreparative HPLC separations were carried out using a system consisting of an NP7000 module (Hanbon Co. Ltd.), a Shodex RID 102 detector (Showa Denko Group), and a Venusil XBP C18 column (8.0 mm × 250 mm, ODS, 5 µm, Bonna-Agela Technologies). Silica gel H (Qingdao marine Chemical), MCI resin (50 µm, Mitsubishi Chemicals), and ODS (Octadecylsilyl) silica gel (120 Å, 50 µm, YMC) were used for column chromatography performance.


#

Plant material

The roots of M. tenacissima were collected from Zhenyuan, Simao, Yunnan province of China and were identified by Prof. Li-Xia Zhang. A voucher specimen (NO.111 010) was deposited in the herbarium of the Beijing Institute of Radiation Medicine, Beijing, China.


#

Extraction and isolation

The dried roots of M. tenacissima (3 kg) were crushed and extracted with 95 % EtOH (24 L) at 120 °C three times (each time for 1 h). The filtrate was concentrated in vacuo to yield an extract and then partitioned between EtOAc and H2O. After concentration, the residue (78 g) of the EtOAc extract was separated on a silica gel column (10 cm × 24 cm) eluted with a gradient consisting of CH3Cl3-MeOH (v/v, 50 : 1 → 7 : 1) to afford 374 fractions (Fr.1 1–374, each fraction 150 ml).

Fr.1 195–214 (6.5 g) was subjected to an MCI resin column (5 cm × 20 cm) eluted with MeOH-H2O to afford 17 fractions (v/v, 70 : 30 for Fr.2 1–13; v/v, 80 : 20 for Fr.2 14–17, each fraction 250 ml). Fr.2 8 (225 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 69 : 31, flow rate 5.0 mL/min) to give compound 6 (12.6 mg, t R 23.2 min), and Fr.2 9 (261 mg) was purified by semipreparative HPLC with CH3OH-H2O (v/v, 70 : 30, flow rate 4.5 mL/min) to yield an additional compound 6 (23.8 mg, t R 25.0 min). Fr.2 14–15 (745 mg) was loaded on an ODS C18 column (2 cm × 30 cm) eluted with CH3OH-H2O (v/v, 67 : 33) to give 16 fractions (Fr.3 1–16, each 50 mL). Then Fr.3 9–11 (293 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 69 : 31, flow rate 4.5 mL/min) to give mixture A (t R 27.0 min), and Fr.3 13–16 (290 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 70 : 30, flow rate 4.5 mL/min) to give mixtures B (t R 20.5 min) and C (t R 22.5 min). Furthermore, mixture A was purified by semipreparative HPLC with CH3CN-H2O (v/v, 45 : 55, flow rate 4.5 mL/min) to yield compounds 5 (66.8 mg, t R 38.0 min) and 3 (24.6 mg, t R 42.5 min). Mixture B was purified by semipreparative HPLC with CH3CN-H2O (v/v, 45 : 55, flow rate 4.5 mL/min) to yield compounds 9 (21.4 mg, t R 39.0 min) and 10 (45.5 mg, t R 42.1 min). Mixture C was purified by semipreparative HPLC with CH3CN-H2O (v/v, 45 : 55, flow rate 5.0 mL/min) to yield compounds 15 (21.4 mg, t R 36.2 min) and 13 (45.5 mg, t R 39.6 min).

Fr.1 300–323 (6.0 g) was subjected to an MCI resin column (5 cm × 20 cm) eluted with MeOH-H2O to afford 30 fractions (v/v, 70 : 30 for Fr.4 1–21; v/v, 75 : 25 for Fr.4 22–30, each fraction 250 mL). Fr.4 16–19 (346.5 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 66 : 34, flow rate 3.5 mL/min) to yield compound 12 (50.1 mg, t R 50.1 min), and mixtures D (21.5 mg, t R 32.3 min) and E (45.4 mg, t R 42.4 min). Fr.4 20–21 (214.4 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 66 : 34, flow rate 3.5 mL/min) to obtain the additional mixtures D (43.1 mg, t R 30.1 min) and E (24.5 mg, t R 40.5 min). Fr.4 22–25 (750 mg) was loaded on an ODS C18 column (2 cm × 30 cm) eluted with CH3OH-H2O (v/v, 64 : 33–70 : 30) to give Fr.5 12–16 (each 50 mL) and Fr.5 17 (MeOH elution). Fr.5 12–16 (239.5 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 69 : 31, flow rate 4.0 mL/min) to afford an additional mixture E (42.6 mg, t R 35.3 min) as well as mixture F (83.8 mg, t R 39.5 min). Fr.5 17 (360 mg) was separated by semipreparative HPLC with CH3OH-H2O (v/v, 70 : 30, flow rate 4.0 mL/min) to give mixture G (76.7 mg, t R 37.3 min). Then, the combined mixture D was purified by semipreparative HPLC with CH3CN-H2O (v/v, 35 : 65, flow rate 4.5 mL/min) to give compounds 14 (13.8 mg, t R 49.7 min) and 11 (26.7 mg, t R 52.0 min). The combined mixture E was purified by semipreparative HPLC with CH3CN-H2O (v/v, 40 : 60, flow rate 4.5 mL/min) to give compound 2 (11.1 mg, t R 41.0 min). Mixture F was purified by semipreparative HPLC with CH3CN-H2O (v/v, 40 : 60, flow rate 4.5 mL/min) to give compounds 4 (45.5 mg, t R 40.6 min) and 1 (15.5 mg, t R 41.4 min). Mixture G was purified by semipreparative HPLC with CH3CN-H2O (v/v, 40 : 60, flow rate 4.0 mL/min) to give compounds 7 (20.5 mg, t R 35.2 min) and 8 (30.0 mg, t R 40.1 min).


#

Characterization

Marstenacisside B10 (1): white amorphous powder; [α]D 20 = + 14.2 (c = 0.059, pyridine). UV (MeOH) λ max 225.8 (ε 16 354) nm. C59H84O24, HRESIMS (positive): m/z 1199.5303 [M + Na]+ (calcd. for C59H84O24Na: 1199.5250). 1H NMR data (600 MHz, pyridine-d 5): δ 3.80 (1 H, m, H-3), 5.97 (1 H, t, J = 10.1 Hz, H-11), 5.59 (1 H, d, J = 10.1 Hz, H-12), 3.11 (1 H, dd, J = 11.2, 6.2 Hz, H-17), 1.55 (3 H, s, 18-CH3), 1.54 (3 H, s, 19-CH3), 1.99 (3 H, s, 21-CH3), 6.77 (1 H, qq, J = 7.1, 1.4 Hz, Tig-H-3), 1.33 (3 H, d, J = 7.1 Hz, Tig-H-4), 1.50 (3 H, s, Tig-H-5), 8.15 (2 H, dd, J = 7.8, 1.3 Hz, Bz-H-3, 7), 7.36 (2 H, dd, J = 7.8, 7.4 Hz, Bz-H-4, 6), 7.46 (1 H, t, J = 7.4 Hz, Bz-H-5), 4.76 (1 H, dd, J = 9.4, 1.5 Hz, Ole-H-1), 1.71 (1 H, m, Ole-H-2a), 2.39 (1 H, m, Ole-H-2b), 3.56 (2 H, overlap, Ole-H-3,5), 3.55 (1 H, overlap, Ole-H-4), 3.53 (1 H, overlap, Ole-H-4), 1.56 (3 H, d, J = 5.3 Hz, Ole-H-6), 3.48 (3 H, s, Ole-3-OCH3), 5.24 (1 H, d, J = 8.2 Hz, Allo-H-1), 3.80 (1 H, overlap, Allo-H-2), 4.39 (1 H, t, J = 2.7 Hz, Allo-H-3), 3.66 (1 H, dd, J = 9.6, 2.4 Hz, Allo-H-4), 4.23 (1 H, m, Allo-H-5), 1.61 (3 H, d, J = 6.2 Hz, Allo-H-6), 3.79 (1 H, s, Allo-3-OCH3), 4.90 (1 H, d, J = 7.8 Hz, Glc1-H-1), 3.99 (1 H, dd, J = 8.2, 7.8 Hz, Glc1-H-2), 4.25 (1 H, m, Glc1-H-3), 4.28 (1 H, m, Glc1-H-4), 3.93 (1 H, m, Glc1-H-5), 4.50 (1 H, m, Glc1-H-6a), 4.43 (1 H, dd, J = 11.6 Hz, Glc1-H-6b), 5.19 (1 H, d, J = 7.9 Hz, Glc2-H-1), 4.10 (1 H, dd, J = 8.2, 7.9 Hz Glc2-H-2), 4.20 (1 H, m, Glc2-H-3), 4.19 (1 H, m, Glc2-H-4), 4.01 (1 H, m, Glc2-H-5), 4.52 (1 H, d, J = 11.4 Hz, Glc2-H-6a), 4.30 (1 H, m, Glc2-H-6b). 13C NMR data (150 MHz, pyridine-d 5), see [Tables 2] and [3].

Table 213C chemical shifts of the aglycones of 115 (150 MHz for 1, 4, 6, 11, and 12, and 125 MHz for 2, 3, 5, 710, and 1315, pyridine-d 5).

Position

1

2

3

4

7

8

9

10

11

12

13

14

15

1

38.8

38.7

38.8

37.7

39.6

39.6

39.6

39.6

38.8

38.8

38.8

38.1

38.3

2

30.0

29.9

29.9

29.7

30.0

29.9

30.0

29.9

30.4

30.4

30.3

30.3

30.3

3

77.0

77.0

76.9

76.0

76.3

76.2

76.3

76.2

77.3

77.3

77.2

76.2

76.1

4

39.6

39.6

39.6

35.0

35.7

35.7

35.7

35.7

39.8

39.8

39.8

35.5

35.4

5

141.5

141.5

141.5

44.2

45.8

45.7

45.7

45.7

139.7

139.8

139.7

44.7

44.7

6

119.6

119.6

119.6

27.4

25.4

25.4

25.4

25.4

122.6

122.5

122.6

29.4

29.4

7

33.1

33.1

33.1

32.5

35.2

35.2

35.2

35.2

28.2

28.3

28.3

28.4

28.4

8

64.3

64.2

64.3

66.3

78.4

78.5

78.4

78.5

37.5

37.6

37.7

40.0

40.3

9

49.7

49.7

49.6

52.5

51.4

51.5

51.4

51.5

47.9

48.1

48.1

50.1

50.3

10

40.4

40.4

40.4

39.7

38.5

38.5

38.5

38.5

39.5

39.5

39.5

37.8

38.0

11

68.7

68.7

68.7

68.5

71.3

72.2

71.3

72.2

71.8

71.9

72.7

71.6

71.7

12

79.9

79.1

79.8

80.1

79.8

78.8

79.8

78.8

78.6

77.7

77.6

79.1

79.1

13

47.4

47.5

47.4

47.5

55.8

55.7

55.8

55.7

54.9

54.9

54.9

55.0

55.0

14

70.9

70.8

70.8

71.7

85.6

85.6

85.6

85.6

84.1

84.2

84.2

83.9

84.0

15

28.3

28.4

28.3

28.2

36.3

36.4

36.3

36.4

34.6

34.8

34.8

33.9

34.0

16

26.5

26.5

26.5

26.4

24.8

24.8

24.8

24.8

24.0

24.1

24.1

24.3

24.4

17

61.4

61.3

61.4

61.3

59.5

59.4

59.5

59.4

58.3

58.3

58.3

58.4

58.4

18

11.9

11.9

11.9

11.9

14.1

14.1

14.1

14.1

11.7

11.7

11.7

11.9

11.9

19

19.2

19.2

19.2

13.1

13.5

13.5

13.5

13.5

19.3

19.3

19.4

12.4

12.4

20

207.8

208.0

207.8

207.8

213.9

214.0

214.0

214.0

213.5

213.7

213.8

213.4

213.4

21

31.3

31.4

31.3

31.2

31.8

31.8

31.8

31.8

31.8

31.8

31.8

31.7

31.7

11-O-

Tig

Tig1

Tig

Tig

Tig

Bz

Tig

Bz

Ac

Tig1

Bz

Ac

Tig

1

167.0

167.0

167.0

167.2

167.1

166.0

167.1

166.0

170.2

167.1

166.1

170.4

167.4

2

128.6

128.9

128.6

128.8

129.0

131.0

129.0

131.0

21.3

129.0

130.8

21.3

128.9

3

138.7

138.6

138.8

138.5

138.6

130.1

138.6

130.1

138.5

130.1

138.6

4

14.1

14.2

14.1

14.1

14.1

128.8

14.1

128.8

14.3

128.8

14.1

5

11.7

12.0

11.7

11.7

11.8

133.3

11.8

133.3

12.0

133.5

11.7

6

128.8

128.8

128.8

7

130.1

130.1

130.1

12-O-

Bz

Tig2

Bz

Bz

Bz

Tig

Bz

Tig

Bz

Tig2

Tig

Bz

Bz

1

166.5

167.5

166.5

166.5

166.9

168.0

166.9

168.0

166.8

167.9

167.8

166.8

166.8

2

130.6

128.8

130.6

130.7

130.4

128.4

130.4

128.4

130.1

128.5

128.3

130.1

130.3

3

130.0

138.4

130.0

130.0

130.1

138.5

130.1

138.5

130.2

138.7

138.8

130.2

130.1

4

128.8

14.3

128.8

128.8

128.9

14.1

128.9

14.1

129.2

14.3

14.2

129.2

128.9

5

133.5

12.0

133.5

133.4

133.6

11.8

133.6

11.8

134.0

12.1

11.9

133.9

133.7

6

128.8

128.8

128.8

128.9

129.0

129.2

129.2

128.9

7

130.0

130.0

130.0

130.1

130.1

130.2

130.2

130.1

Table 313C chemical shifts of the sugar moieties of 115 (150 MHz for 1, 4, 6, 11, and 12, and 125 MHz for 2, 3, 5, 710, and 1315, pyridine-d 5).

Position

1

2

3

4

7

8

9

10

11

12

13

14

15

Ole-1

97.9

97.9

97.9

97.5

97.5

97.4

97.5

97.4

98.0

98.0

97.9

97.7

97.5

− 2

37.7

37.7

37.8

37.8

37.8

37.8

37.8

37.8

37.8

37.8

37.8

37.8

37.8

− 3

79.6

79.6

79.6

79.6

79.6

79.6

79.8

79.6

79.6

79.6

79.6

79.7

79.6

− 4

83.2

83.2

83.2

83.2

83.3

83.2

83.3

83.2

83.3

83.2

83.2

83.3

83.3

− 5

71.9

71.9

71.9

71.9

71.9

71.9

72.0

71.9

71.9

71.9

71.9

72.0

71.9

− 6

19.0

19.0

19.0

19.0

19.0

19.0

19.1

19.1

19.0

19.0

19.0

19.1

19.0

3-OCH3

57.2

57.2

57.2

57.2

57.2

57.1

57.2

57.2

57.2

57.2

57.2

57.2

57.3

Allo-1

101.9

101.9

101.9

101.9

101.9

101.9

101.9

101.9

101.9

101.9

101.9

101.9

101.9

− 2

72.7

72.7

72.7

72.7

72.7

72.7

72.7

72.7

72.7

72.7

72.8

72.7

72.7

− 3

83.1

83.1

83.2

83.1

83.1

83.1

83.2

83.2

83.1

83.1

83.2

83.1

83.2

− 4

83.4

83.4

83.3

83.4

83.4

83.4

83.4

83.3

83.4

83.4

83.3

83.4

83.3

− 5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

69.5

− 6

18.3

18.3

18.3

18.3

18.3

18.3

18.3

18.3

18.3

18.3

18.3

18.3

18.3

3-OCH3

61.7

61.7

61.7

61.7

61.7

61.7

61.7

61.7

61.7

61.7

61.7

61.7

61.7

Glc1-1

106.6

106.6

106.6

106.2

106.2

106.2

106.6

106.6

106.2

106.2

106.6

106.2

106.6

− 2

75.0

75.0

75.5

75.0

75.0

75.0

75.5

75.5

75.0

75.0

75.5

75.0

75.5

− 3

76.6

76.6

78.4

76.6

76.6

76.6

78.4

78.4

76.6

76.6

78.4

76.6

78.4

− 4

81.5

81.5

71.9

81.5

81.5

81.5

72.0

72.0

81.5

81.5

71.9

81.5

71.9

− 5

76.4

76.4

78.4

76.4

76.4

76.4

78.4

78.4

76.4

76.4

78.4

76.4

78.4

− 6

63.4

62.4

63.0

62.5

62.5

62.4

63.0

63.0

62.4

62.4

63.0

62.4

63.0

Glc2-1

105.0

105.0

105.0

105.0

105.0

105.0

105.0

105.0

− 2

74.8

74.8

74.8

74.8

74.8

74.8

74.8

74.8

− 3

78.3

78.3

78.3

78.3

78.3

78.3

78.3

78.3

− 4

71.6

71.6

71.6

71.6

71.5

71.5

71.6

71.6

− 5

78.6

78.6

78.6

78.6

78.6

78.6

78.6

78.6

− 6

62.5

62.5

62.5

62.4

62.5

62.5

62.5

62.5

Marstenacisside B11 (2): white amorphous powder; [α]D 20 = + 23.7 (c = 0.038, pyridine). UV (MeOH) λ max 215.6 (ε 14 112) nm. C57H86O24, HRESIMS (positive): m/z 1177.5454 [M + Na]+ (calcd. for C57H86O24Na: 1177.5407). 1H NMR data (500 MHz, pyridine-d 5): δ 3.79 (1 H, m, H-3), 5.88 (1 H, t, J = 10.2 Hz, H-11), 5.44 (1 H, d, J = 10.2 Hz, H-12), 3.06 (1 H, dd, J = 11.2, 6.2 Hz, H-17), 1.49 (3 H, s, 18-CH3), 1.54 (3 H, s, 19-CH3), 2.10 (3 H, s, 21-CH3), 6.95 (1 H, qq, J = 7.1, 1.3 Hz, Tig1-H-3), 1.57 (3 H, d, J = 7.1 Hz, Tig1-H-4), 1.78 (3 H, s, Tig1-H-5), 6.93 (1 H, qq, J = 7.1, 1.3 Hz, Tig2-H-3), 1.57 (3 H, d, J = 7.1 Hz, Tig2-H-4), 1.84 (3 H, s, Tig2-H-5), 4.77 (1 H, d, J = 9.7 Hz, Ole-H-1), 3.50 (3 H, s, Ole-3-OCH3), 5.27 (1 H, d, J = 8.1 Hz, Allo-H-1), 3.81 (1 H, s, Allo-3-OCH3), 4.92 (1 H, d, J = 7.8 Hz, Glc1-H-1), 5.22 (1 H, d, J = 7.9 Hz, Glc2-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside A8 (3): white amorphous powder; [α]D 20 = + 10.8 (c = 0.051, pyridine). UV (MeOH) λ max 225.6 (ε 22 516) nm. C53H74O19, HRESIMS (negative): m/z 1013.4787 [M – H] (calcd. for C53H73O19: 1013.4746). 1H NMR data (500 MHz, pyridine-d 5): δ 3.81 (1 H, m, H-3), 6.00 (1 H, t, J = 10.1 Hz, H-11), 5.62 (1 H, d, J = 10.1 Hz, H-12), 3.11 (1 H, dd, J = 11.5, 6.2 Hz, H-17), 1.57 (3 H, s, 18-CH3), 1.56 (3 H, s, 19-CH3), 2.01 (3 H, s, 21-CH3), 6.79 (1 H, q, J = 7.1 Hz, Tig-H-3), 1.35 (3 H, d, J = 7.0 Hz, Tig-H-4), 1.52 (3 H, s, Tig-H-5), 8.18 (2 H, d, J = 7.9 Hz, Bz-H-3, 7), 7.38 (2 H, dd, J = 7.9, 7.4 Hz, Bz-H-4, 6), 7.48 (1 H, t, J = 7.4 Hz, Bz-H-5), 4.78 (1 H, d, J = 9.4 Hz, Ole-H-1), 1.74 (1 H, m, Ole-H-2a), 2.46 (1 H, m, Ole-H-2b), 3.58 (2 H, overlap, Ole-H-3, 5), 3.57 (1 H, overlap, Ole-H-4), 3.55 (1 H, overlap, Ole-H-4), 1.59 (3 H, d, J = 4.9 Hz, Ole-H-6), 3.51 (3 H, s, Ole-3-OCH3), 5.27 (1 H, d, J = 8.0 Hz, Allo-H-1), 3.81 (1 H, overlap, Allo-H-2), 4.48 (1 H, br s, Allo-H-3), 3.75 (1 H, dd, J = 9.6, 2.0 Hz, Allo-H-4), 4.27 (1 H, m, Allo-H-5), 1.61 (3 H, d, J = 6.1, Allo-H-6), 3.83 (1 H, s, Allo-3-OCH3), 4.99 (1 H, d, J = 7.7 Hz, Glc1-H-1), 4.04 (1 H, dd, J = 8.2, 7.8 Hz, Glc-H-2), 4.25 (1 H, m, Glc-H-3), 4.23 (1 H, m, Glc-H-4), 4.00 (1 H, m, Glc-H-5), 4.55 (1 H, m, Glc-H-6a), 4.39 (1 H, br d, J = 11.6 Hz, Glc-H-6b). 13C NMR (125 MHz, pyridine-d 5), data see [Tables 2] and [3].

Marstenacisside B12 (4): white amorphous powder; [α]D 20 = + 34.4 (c = 0.0465, pyridine). UV (MeOH) λ max 225.0 (ε 13 750) nm. C59H86O24, HRESIMS (negative): m/z 1177.5488 [M – H] (calcd. for C59H85O24: 1177.5431). 1H NMR data (600 MHz, pyridine-d 5): δ 3.84 (1 H, m, H-3), 5.84 (1 H, t, J = 10.2 Hz, H-11), 5.52 (1 H, d, J = 9.9 Hz, H-12), 3.07 (1 H, dd, J = 11.7, 6.3 Hz, H-17), 1.53 (3 H, s, 18-CH3), 1.26 (3 H, s, 19-CH3), 2.00 (3 H, s, 21-CH3), 6.74 (1 H, qq, J = 7.1, 1.2 Hz, Tig-H-3), 1.34 (3 H, d, J = 7.1 Hz, Tig-H-4), 1.52 (3 H, s, Tig-H-5), 8.14 (2 H, dd, J = 8.0, 1.2 Hz, Bz-H-3, 7), 7.35 (2 H, dd, J = 7.8, 7.2 Hz, Bz-H-4, 6), 7.46 (1 H, t, J = 7.2 Hz, Bz-H-5), 4.77 (1 H, br d, J = 9.2 Hz, Ole-H-1), 3.49 (3 H, s, Ole-3-OCH3), 5.26 (1 H, d, J = 8.1 Hz, Allo-H-1), 3.79 (1 H, s, Allo-3-OCH3), 4.92 (1 H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1 H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside B13 (7): white amorphous powder; [α]D 25 = + 35.8 (c = 0.0608, MeOH). UV (MeOH) λ max 225.4 (ε 13 167) nm. C59H88O25, HRESIMS (negative): m/z 1195.5596 [M – H] (calcd. for C59H87O25: 1195.5536). 1H NMR data (500 MHz, pyridine-d 5): δ 3.91 (1 H, m, H-3), 6.61 (1 H, t, J = 10.6 Hz, H-11), 5.60 (1 H, d, J = 10.0 Hz, H-12), 3.35 (1 H, dd, J = 9.0, 5.2 Hz, H-17), 1.72 (3 H, s, 18-CH3), 1.53 (3 H, s, 19-CH3), 2.06 (3 H, s, 21-CH3), 6.82 (1 H, q, J = 7.1 Hz, Tig-H-3), 1.38 (3 H, d, J = 7.1 Hz, Tig-H-4), 1.54 (3 H, s, Tig-H-5), 8.26 (2 H, d, J = 7.6 Hz Bz-H-3, 7), 7.42 (2 H, t, J = 7.7 Hz, Bz-H-4, 6), 7.51 (1 H, t, J = 7.4 Hz, Bz-H-5), 4.81 (1 H, br d, J = 9.6 Hz, Ole-H-1), 3.49 (3 H, s, Ole-3-OCH3), 5.28 (1 H, d, J = 8.0 Hz, Allo-H-1), 3.81 (1 H, s, Allo-3-OCH3), 4.92 (1 H, d, J = 7.7 Hz, Glc1-H-1), 5.21 (1 H, d, J = 7.9 Hz, Glc2-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside B14 (8): white amorphous powder; [α]D 25 = + 29.7 (c = 0.0738, MeOH). UV (MeOH) λ max 225.4 (ε 19 513) nm. C59H88O25, HRESIMS (negative): m/z 1195.5588 [M – H] (calcd. for C59H87O25: 1195.5536). 1H NMR data (500 MHz, pyridine-d 5): δ 3.86 (1 H, m, H-3), 6.67 (1 H, t, J = 10.6 Hz, H-11), 5.55 (1 H, d, J = 10.0 Hz, H-12), 3.30 (1 H, dd, J = 9.0, 5.2 Hz, H-17), 1.68 (3 H, s, 18-CH3), 1.54 (3 H, s, 19-CH3), 2.16 (3 H, s, 21-CH3), 8.19 (2 H, d, J = 7.5 Hz Bz-H-3, 7), 7.40 (2 H, dd, J = 7.7, 7.2 Hz, Bz-H-4, 6), 7.47 (1 H, t, J = 7.2 Hz, Bz-H-5), 6.88 (1 H, q, J = 7.0 Hz, Tig-H-3), 1.40 (3 H, d, J = 7.0 Hz, Tig-H-4), 1.60 (3 H, s, Tig-H-5), 4.78 (1 H, d, J = 9.5 Hz, Ole-H-1), 3.48 (3 H, s, Ole-3-OCH3), 5.27 (1 H, d, J = 7.9 Hz, Allo-H-1), 3.80 (1 H, s, Allo-3-OCH3), 4.92 (1 H, d, J = 7.6 Hz, Glc1-H-1), 5.21 (1 H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside A9 (9): white amorphous powder; [α]D 25 = + 47.0 (c = 0.0885, MeOH). UV (MeOH) λ max 225.0 (ε 15 189) nm. C53H78O20, HRESIMS (negative): m/z 1033.5083 [M – H] (calcd. for C53H77O20: 1195.5048). 1H NMR data (500 MHz, pyridine-d 5): δ 3.91 (1 H, m, H-3), 6.61 (1 H, t, J = 10.6 Hz, H-11), 5.60 (1 H, d, J = 10.1 Hz, H-12), 3.35 (1 H, dd, J = 9.0, 5.2 Hz, H-17), 1.72 (3 H, s, 18-CH3), 1.53 (3 H, s, 19-CH3), 2.06 (3 H, s, 21-CH3), 6.82 (1 H, q, J = 7.1 Hz, Tig-H-3), 1.38 (3 H, d, J = 7.1 Hz, Tig-H-4), 1.54 (3 H, s, Tig-H-5), 8.26 (2 H, d, J = 7.6 Hz, Bz-H-3, 7), 7.42 (2 H, t, J = 7.7 Hz, Bz-H-4, 6), 7.51 (1 H, t, J = 7.4 Hz, Bz-H-5), 4.82 (1 H, d, J = 9.7 Hz, Ole-H-1), 3.50 (3 H, s, Ole-3-OCH3), 5.29 (1 H, d, J = 8.1 Hz, Allo-H-1), 3.83 (1 H, s, Allo-3-OCH3), 4.99 (1 H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside A10 (10): white amorphous powder; [α]D 25 = + 35.3 (c = 0.0962, MeOH). UV (MeOH) λ max 225.2 (ε 14 777) nm. C53H78O20, HRESIMS (negative): m/z 1033.5059 [M – H] (calcd. for C53H77O20: 1033.5048). 1H NMR data (500 MHz, pyridine-d 5): δ 3.87 (1 H, m, H-3), 6.67 (1 H, t, J = 10.7 Hz, H-11), 5.55 (1 H, d, J = 10.1 Hz, H-12), 3.29 (1 H, dd, J = 9.2, 5.4 Hz, H-17), 1.68 (3 H, s, 18-CH3), 1.54 (3 H, s, 19-CH3), 2.16 (3 H, s, 21-CH3), 8.19 (2 H, d, J = 7.5 Hz, Bz-H-3, 7), 7.41 (2 H, dd, J = 7.5, 7.3 Hz, Bz-H-4, 6), 7.47 (1 H, t, J = 7.3 Hz, Bz-H-5), 6.88 (1 H, q, J = 7.0 Hz, Tig-H-3), 1.40 (3 H, d, J = 7.0 Hz, Tig-H-4), 1.60 (3 H, s, Tig-H-5), 4.79 (1 H, d, J = 9.5 Hz, Ole-H-1), 3.48 (3 H, s, Ole-3-OCH3), 5.27 (1 H, d, J = 8.2 Hz, Allo-H-1), 3.83 (1 H, s, Allo-3-OCH3), 4.98 (1 H, d, J = 7.8 Hz, Glc-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside B15 (11): white amorphous powder; [α]D 25 = + 7.0 (c = 0.0938, MeOH). UV (MeOH) λ max 230.4 (ε 9408) nm. C56H82O24, HRESIMS (negative): m/z 1137.5137 [M – H] (calcd. for C56H81O24: 1137.5118). 1H NMR data (600 MHz, pyridine-d 5): δ 3.77 (1 H, m, H-3), 5.85 (1 H, t, J = 10.2 Hz, H-11), 5.43 (1 H, d, J = 10.2 Hz, H-12), 3.23 (1 H, dd, J = 9.6, 4.8 Hz, H-17), 1.44 (3 H, s, 18-CH3), 1.25 (3 H, s, 19-CH3), 2.06 (3 H, s, 21-CH3), 8.36 (2 H, d, J = 7.9 Hz, Bz-H-3, 7), 7.49 (2 H, dd, J = 7.9, 7.4 Hz, Bz-H-4, 6), 7.57 (1 H, overlap, Bz-H-5), 4.75 (1 H, d, J = 9.2 Hz, Ole-H-1), 3.49 (3 H, s, Ole-3-OCH3), 5.26 (1 H, d, J = 8.0 Hz, Allo-H-1), 3.79 (1 H, s, Allo-3-OCH3), 4.90 (1 H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1 H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data (150 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside B16 (12): white amorphous powder; [α]D 25 = + 41.6 (c = 0.0608, MeOH). UV (MeOH) λ max 215.2 (ε 17 679) nm. C57H88O24, HRESIMS (negative): m/z 1155.5636 [M – H] (calcd. for C57H87O24: 1155.5587). 1H NMR data (600 MHz, pyridine-d 5): δ 3.76 (1 H, m, H-3), 5.88 (1 H, t, J = 10.4 Hz, H-11), 5.29 (1 H, d, J = 10.0 Hz, H-12), 3.18 (1 H, dd, J = 9.3, 4.8 Hz, H-17), 1.38 (3 H, s, 18-CH3), 1.31 (3 H, s, 19-CH3), 2.17 (3 H, s, 21-CH3), 6.92 (1 H, qq, J = 7.1, 1.4 Hz, Tig1-H-3), 1.57 (3 H, d, J = 7.1 Hz, Tig1-H-4), 1.78 (3 H, s, Tig1-H-5), 7.06 (1 H, qq, J = 7.1, 1.3 Hz, Tig2-H-3), 1.64 (3 H, d, J = 7.1 Hz, Tig2-H-4), 1.89 (3 H, s, Tig2-H-5), 4.74 (1 H, br d, J = 9.2 Hz, Ole-H-1), 3.48 (3 H, s, Ole-3-OCH3), 5.24 (1 H, d, J = 8.1 Hz, Allo-H-1), 3.79 (1 H, s, Allo-3-OCH3), 4.90 (1 H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1 H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data (150 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside A11 (13): white amorphous powder; [α]D 25 = + 51.7 (c = 0.0769, MeOH). UV (MeOH) λ max 225.8 (ε 14 318) nm. C53H76O19, HRESIMS (negative): m/z 1015.4949 [M – H] (calcd. for C53H75O19: 1015.4903). 1H NMR data (500 MHz, pyridine-d 5): δ 3.75 (1 H, m, H-3), 6.08 (1 H, t, J = 10.2 Hz, H-11), 5.44 (1 H, d, J = 10.0 Hz, H-12), 3.22 (1 H, dd, J = 9.4, 4.7 Hz, H-17), 1.37 (3 H, s, 18-CH3), 1.25 (3 H, s, 19-CH3), 2.18 (3 H, s, 21-CH3), 8.16 (2 H, d, J = 7.8 Hz Bz-H-3, 7), 7.38 (2 H, dd, J = 7.8, 7.4 Hz, Bz-H-4, 6), 7.48 (1 H, J = 7.6, Bz-H-5), 6.92 (1 H, q, J = 7.0 Hz, Tig-H-3), 1.46 (3 H, d, J = 7.0 Hz, Tig-H-4), 1.66 (3 H, s, Tig-H-5), 4.72 (1 H, br d, J = 9.5 Hz, Ole-H-1), 3.50 (3 H, s, Ole-3-OCH3), 5.25 (1 H, d, J = 8.0 Hz, Allo-H-1), 3.82 (1 H, s, Allo-3-OCH3), 4.98 (1 H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside B17 (14): white amorphous powder; [α]D 25 = + 8.2 (c = 0.0500, MeOH). UV (MeOH) λ max 230.4 (ε 16 817) nm. C56H84O24, HRESIMS (negative): m/z 1139.5333 [M – H] (calcd. for C56H83O24: 1139.5274). 1H NMR data (500 MHz, pyridine-d 5): δ 3.82 (1 H, m, H-3), 5.69 (1 H, t, J = 10.2 Hz, H-11), 5.36 (1 H, d, J = 9.8 Hz, H-12), 3.22 (1 H, dd, J = 8.8, 5.0 Hz, H-17), 1.41 (3 H, s, 18-CH3), 0.99 (3 H, s, 19-CH3), 2.05 (3 H, s, 21-CH3), 8.35 (2 H, d, J = 7.9 Hz, Bz-H-3, 7), 7.49 (2 H, dd, J = 7.9, 7.4 Hz, Bz-H-4, 6), 7.57 (1 H, overlap, Bz-H-5), 4.77 (1 H, br d, J = 9.2 Hz, Ole-H-1), 3.50 (3 H, s, Ole-3-OCH3), 5.27 (1 H, d, J = 8.1 Hz, Allo-H-1), 3.79 (1 H, s, Allo-3-OCH3), 4.90 (1 H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1 H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].

Marstenacisside A12 (15): white amorphous powder; [α]D 25 = + 37.6 (c = 0.0792, MeOH). UV (MeOH) λ max 225.2 (ε 20 064) nm. C53H78O19, HRESIMS (negative): m/z 1017.5085 [M – H] (calcd. for C53H77O19: 1017.5059). 1H NMR data (500 MHz, pyridine-d 5): δ 3.82 (1 H, m, H-3), 5.87 (1 H, t, J = 10.4 Hz, H-11), 5.43 (1 H, d, J = 9.9 Hz, H-12), 3.28 (1 H, dd, J = 9.1, 5.0 Hz, H-17), 1.46 (3 H, s, 18-CH3), 1.07 (3 H, s, 19-CH3), 2.08 (3 H, s, 21-CH3), 6.78 (1 H, q, J = 7.1 Hz, Tig-H-3), 1.40 (3 H, d, J = 7.1 Hz, Tig-H-4), 1.54 (3 H, s, Tig-H-5), 8.27 (2 H, d, J = 7.7 Hz, Bz-H-3, 7), 7.45 (2 H, dd, J = 7.7, 7.5 Hz, Bz-H-4, 6), 7.53 (1 H, t, J = 7.4 Hz, Bz-H-5), 4.77 (1 H, br d, J = 9.7 Hz, Ole-H-1), 3.50 (3 H, s, Ole-3-OCH3), 5.27 (1 H, d, J = 8.0 Hz, Allo-H-1), 3.80 (1 H, s, Allo-3-OCH3), 4.98 (1 H, d, J = 7.8 Hz, Glc-H-1). 13C NMR data (125 MHz, pyridine-d 5), see [Tables 2] and [3].


#

HIV inhibition assay

SupT1 cells (2 × 105) were co-transfected with 0.6 mg of pNL-Luc-E and 0.4 mg of pHIT/G. Then the VSV-G pseudo-typed viral supernatant (HIV-1) was harvested by filtration through a 0.45-µm filter after 48 h and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudo-typed HIV-1 (MOI = 1) at 37.8 °C for 48 h in the absence or presence of the test compounds (with the positive control, Efavirenz) [14]. A luciferase assay system (Promega) was used to determine the inhibition rate. The cytotoxicity was measured by the MTT method. SupT1 cells were seeded into a 96-well microtiter plate in the absence or presence of the test compounds (positive control, Efavirenz) in triplicate and incubated at 37.8 °C in a humid atmosphere of 5 % CO2. After a 4-day incubation, cell viability was measured by the MTT method. Purities of all of the tested compounds were > 95 %, as detected by HPLC-ELSD. Efavirenz were obtained from NIH-AIDS Research and Reference Reagent Program with a purity of > 98 % (HPLC)


#

Supporting information

NMR spectra of 115 are available as Supporting Information.


#
#

Acknowledgements

Thanks for the financial support from the National Natural Science Foundation of China (NSFC) No. 31170041 and National Infrastructure of Microbial Resources (No. NIMR-2015-3). Thanks to Miss. Meifeng Xu in the National Center of Biomedical Analysis (NCBA) for the measurements of the NMR spectra.


#
#

Conflict of Interest

There are no conflicts of interest for all authors.

* These authors contributed equally to this work.


Supporting Information


Correspondence

Prof. Baiping Ma
Beijing Institute of Radiation Medicine
No. 27 Tai-ping Road, Haidian District
Beijing 100850
Peopleʼs Republic of China
Phone: +86 10 68 21 00 77   
Fax: +86 10 68 21 46 53   

 

Prof. Lixia Zhang
Yunnan Branch of Institute of Medicinal Plant Development
Chinese Academy of Medical Sciences & Peking Union Medical College
NO. 138 Xuanwei Street
Jinghong 666100
China


Zoom Image
Fig. 1 Structures of 115.
Zoom Image
Fig. 2 Key HMBC and 1H-1H COSY correlations for 1.
Zoom Image
Fig. 3 Key ROESY correlations of the aglycone for 1.