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

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.

Key words

Marsdenia tenacissima - Asclepiadaceae - polyoxypregnane glycoside - marstenacisside - anti-HIV - antiviral activity

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 (1–4 and 7–15) and 2 known polyoxypregnane glycosides (5 and 6; [Fig. 1]).

Fig. 1 Structures of 1–15.

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 (1–4 and 7–15) 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.

Fig. 2 Key HMBC and ¹H-¹H COSY correlations for 1.

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 1–15 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 2  ¹³C chemical shifts of the aglycones of 1–15 (150 MHz for 1, 4, 6, 11, and 12, and 125 MHz for 2, 3, 5, 7–10, and 13–15, 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 3  ¹³C chemical shifts of the sugar moieties of 1–15 (150 MHz for 1, 4, 6, 11, and 12, and 125 MHz for 2, 3, 5, 7–10, and 13–15, 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 1–15 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.

• References

• 1 Li HT, Kang LP, Guo BL, Zhang ZL, Guan YH, Pang X, Peng CZ, Ma BP, Zhang LX. Original plant identification of Dai nationality herb “Daibaijie”. Chin J Chin Mater Med 2014; 39: 1525-1529
• 2 Pang X, Kang LP, Yu HS, Zhao Y, Han LF, Zhang J, Xiong CQ, Zhang LX, Yu LY, Ma BP. New polyoxypregnane glycosides from the roots of Marsdenia tenacissima . Steroids 2015; 93: 68-76
• 3 Liang XW, Wang CC. [Analysis of the relation of clearing heat and expelling miasma medicine and antivirus medicine]. J Liaoning Univ Tradit Chin Med 2009; 11: 28-29
• 4 Huang XD, Liu T, Wang S. Two new polyoxypregnane glycosides from Marsdenia tenacissima . Helv Chim Acta 2009; 92: 2111-2117
• 5 Ma BX, Fang TZ, Ma K, Ni J, Wu H, Ding WP, Jiang C. Novel saponins hainaneosides A and B isolated from Marsdenia hainanensis . J Nat Prod 1997; 60: 134-138
• 6 Li QF, Wang XL, Ding LS, Zhang C. Polyoxypregnanes from the stems of Marsdenia tenacissima . Chin Chem Lett 2007; 18: 831-834
• 7 Liu J, Yu ZB, Ye YH, Zhou YW. A new C₂₁ steroid glycoside from Marsdenia tenacissima . Chin Chem Lett 2008; 19: 444-446
• 8 Wang XL, Peng SL, Ding LS. Further polyoxypregnane glycosides from Marsdenia tenacissima . J Asian Nat Prod Res 2010; 12: 654-661
• 9 Deng J, Liao ZX, Chen DF. Marsdenosides A–H, polyoxypregnane glycosides from Marsdenia tenacissima . Phytochemistry 2005; 66: 1040-1051
• 10 Deng J, Liao ZX, Chen DF. Two new C₂₁ steroids from Marsdenia tenacissima . Chin Chem Lett 2005; 16: 487-490
• 11 Wang XL, Li QF, Yu KB, Peng SL, Zhou Y, Ding LS. Four new pregnane glycosides from the stems of Marsdenia tenacissima . Helv Chim Acta 2006; 89: 2738-2744
• 12 Umehara K, Endoh M, Miyase T, Kuroyanagi M, Ueno A. Studies on differentiation inducers. IV. Pregnane derivatives from condurango cortex . Chem Pharm Bull (Tokyo) 1994; 42: 611-616
• 13 Sahu N, Panda N, Mandal N, Banerjee S, Koike K, Nikaido T. Polyoxypregnane glycosides from the flowers of Dregea volubilis . Phytochemistry 2002; 61: 383-388
• 14 Zhang Q, Liu ZL, Mi ZY, Li XY, Jia PP, Zhou JM, Yin X, You XF, Yu LY, Guo F, Ma J, Liang C, Cen S. High-throughput assay to identify inhibitors of Vpu-mediated down-regulation of cell surface BST-2. Antiviral Res 2011; 91: 321-329