New polyoxypregnane glycosides from the roots of Marsdenia tenacissima

Steroids 93 (2015) 68–76

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

New polyoxypregnane glycosides from the roots of Marsdenia tenacissima Xu Pang a,b, Li-Ping Kang c,d,⇑, He-Shui Yu d, Yang Zhao a, Li-Feng Han d, Jie Zhang a, Cheng-Qi Xiong a, Li-Xia Zhang e, Li-Yan Yu b, Bai-Ping Ma a,⇑ a

Beijing Institute of Radiation Medicine, Beijing 100850, China Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China d Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China e Yunnan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Jinghong 666100, China b c

a r t i c l e

i n f o

Article history: Received 14 April 2014 Received in revised form 7 October 2014 Accepted 12 November 2014 Available online 28 November 2014 Keywords: Marsdenia tenacissima Asclepiadaceae Polyoxypregnane glycosides Marstenacissides

a b s t r a c t For the first time, a systematic phytochemical study was performed on the roots of Marsdenia tenacissima. Finally, sixteen new polyoxypregnane glycosides, marstenacissides A1–A7 (1–7) and marstenacissides B1–B9 (8–16), were isolated from M. tenacissima roots. The structures of these new compounds were established by various spectroscopic techniques, including 1D and 2D NMR spectroscopy and mass spectrometry. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Marsdenia tenacissima (Asclepiadaceae) is a perennial climber extensively distributed in Yunnan province of China. The roots of M. tenacissima are widely used as a traditional Dai nationality herb medicine Dai-Bai-Jie by the Dai people living in Laos, Burma and China, especially in Yunnan province of China, due to their functions of detoxification, decreasing swelling and alleviating pain [1]. In addition to its use as a traditional herb medicine, these roots were also used as the main medicinal materials for preparing a series of preparations such as Ya-jie tablets and Bai-jie capsules in China. Previous phytochemical investigations focusing on the stems of M. tenacissima had resulted in the isolation of a series of polyoxypregnane glycosides [2–13], while the works on the roots of this plant had been hardly reported so far. Therefore, for the purpose of investigation on the constituents from the roots of M. tenacissima, a systematic phytochemical study was carried out on ⇑ Corresponding authors at: State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. Tel.: +86 10 64014411x2836 (L.-P. Kang). Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850, China. Tel.: +86 10 68210077x930265; fax: +86 10 68214653 (B.-P. Ma). E-mail addresses: [email protected] (L.-P. Kang), [email protected] (B.-P. Ma). http://dx.doi.org/10.1016/j.steroids.2014.11.004 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

their ethanol extract. Sixteen new polyoxypregnane glycosides (1–16) were isolated by a series of purification steps, and their structures were identified using the spectrographic techniques of HRESIMS and NMR (Fig. 1). This paper mainly presented the isolation and structure elucidation of these new compounds.

2. Experimental 2.1. General methods The HRESIMS was recorded on a Synapt MS (Waters Corporation, Milford, MA, USA). The NMR spectra were performed on Bruker DRX-500 spectrometer (500 MHz for 1H NMR and 125 MHz for 13 C NMR, Karlsruhe, Germany) and Varian UNITYINOVA 600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR, Palo Alto, CA, USA) in pyridine-d5 (Sigma–Aldrich, St. Louis, MO, USA), and the chemical shifts were given in d (ppm). The optical rotations were measured with a JASCO J-810 polarimeter (JASCO Corporation, Tokyo, Japan). HPLC analyses were performed on Agilent 1100 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Techmate C18 column (4.6 mm  250 mm, ODS, 5 lm, Techmate Co. Ltd., Beijing, China) and an Alltech 2000 evaporative light scattering detector (Temp: 110 °C, Gas: 2.4 L/min, Alltech Corporation, Deerfield, USA). Semi-preparative HPLC separations were

69

X. Pang et al. / Steroids 93 (2015) 68–76

performed on the NP7000 module (Hanbon Co. Ltd., Huaian, China.) equipped with a Shodex RID 102 detector (Showa Denko Group, Tokyo, Japan), a Venusil XBP C18 column (8.0 mm  250 mm, ODS, 5 lm, Bonna-Agela, Tianjin, China) and a Megres C18 column (8.0 mm  250 mm, ODS, 5 lm, Hanbon Co. Ltd., Huaian, China). TLC was performed on silica gel GF254 plates (Qingdao marine Chemical, Qingdao, China). Silica gel H (Qingdao marine Chemical, Qingdao, China), MCI gel (50 lm, Mitsubishi Chemicals, Tokyo, Japan) and ODS (Octadecylsilyl) silica gel (120 Å, 50 lm, YMC, Japan) were used for the performance of column chromatography.

Combined mixture A (total 160.6 mg) was purified by semi-preparative HPLC with CH3CN–H2O (v/v, 35:65, flow rate 4.5 mL/ min) to yield compounds 1 (60.7 mg, tR 44.3 min) and 2 (50.9 mg, tR 45.2 min). Fr.2 9 (261 mg) was purified by semi-preparative HPLC with CH3OH–H2O (v/v, 70:30, flow rate 4.5 mL/min) to yield compounds 3 (9.8 mg, tR 30.5 min), 4 (14.5 mg, tR 35.5 min), 5 (9.4 mg, tR 37.7 min) and 6 (4.5 mg, tR 39.2 min). Fr.2 10–11 (515 mg) was loaded on ODS (Octadecylsilyl) silica gel 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 12–14 and Fr.3 15–16 were purified by semi-preparative HPLC with CH3OH–H2O (v/v, 70:30, flow rate 4.5 mL/min) also to yield additional compounds 4 (13.0 mg, tR 34.3 min), 5 (13.5 mg, tR 36.5 min) and 6 (17.3 mg, tR 39.0 min). Fr.2 14–15 (745 mg) was loaded on ODS silica gel column (2 cm  30 cm) eluted with CH3OH–H2O (v/v, 67:33) to give Fr.4 1–16 (each 50 mL), then Fr.4 13–16 was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 70:30, flow rate 4.5 mL/min) to give mixture B (290 mg, tR 20.5 min), and the further purification of mixture B by semi-preparative HPLC with CH3CN–H2O (v/v, 45:55, flow rate 5.0 mL/min) yielded compound 7 (15.7 mg, tR 36.5 min). Fr.1 300–323 (6.0 g) was subjected to a MCI resin column (5 cm  20 cm) eluted with MeOH–H2O to afford 30 fractions (v/v, 70:30 for Fr.5 1–21; v/v, 75:25 for Fr.5 22–30, each fraction 250 mL). Fr.5 10–13 (374.4 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 67:33, flow rate 4.5 mL/min) to give compound 16 (19.0 mg, tR 20.5 min) and mixture C (112.7 mg, tR 16.1 min), then mixture C was further purified by semi-preparative HPLC with CH3CN–H2O (v/v, 35:65, flow rate 4.5 mL/min) to yield compounds 9 (31.5 mg, 44.9 min) and 10 (51.5 mg, tR 47.3 min). Fr.5 14–15 (269.3 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 66:34, flow rate 3.5 mL/min) to yield compound 8 (5.5 mg, tR 36.9 min). Fr.5 16–19 (346.5 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 66:34, flow rate 3.5 mL/min) to yield compounds 8 (8.7 mg, tR 39.0 min) and 12 (36.5 mg, tR 48.3 min) as well as mixture D (45.5 mg, tR 42.1 min). Fr.5 20–21 (214.4 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 66:34, flow rate 3.5 mL/min) to obtain an additional mixture D (24.5 mg, tR 40.5 min). Fr.5 22–25

2.2. Plant material The roots of M. tenacissima were collected from Zhenyuan, Simao of Yunnan province, China, and were identified by Prof. LiXia Zhang. A voucher specimen (No. 111010) was deposited in author’s laboratory in Beijing Institute of Radiation Medicine, Beijing. 2.3. Extraction and isolation The dried roots of M. tenacissima (3 kg) were crushed and extracted with 95% EtOH at 120 °C for three times (24 L, each for 1 h). The filtered solution was concentrated in vacuo to yield a residue. The residue was partitioned between EtOAc (1.5 L) and H2O (1.5 L). After concentration of the EtOAc extract in vacuo, the pasty residue (78 g) was subjected to a silica gel column (10 cm  24 cm) eluted with a gradient mobile phase consisting of CHCl3–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 a MCI gel 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 7 (198 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 69:31, flow rate 4.0 mL/min) to give mixture A (101.4 mg, tR 18.7 min), and Fr.2 8 (225 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 69:31, flow rate 5.0 mL/min) to give another mixture A (59.2 mg, tR 18.1 min).

21

OR 4 R 3O 1 2

11 19 9

10 5

R 1O

H

17

R2 8

13

16

14

15

OH

R 1=S1 R 1=S1 R 1=S1 R 1=S1 R 1=S2 R 1 =S2 R 1 =S2 R 1 =S2

R2 =H R2 =OH R2 =H R2 =H R2 =H R2 =H R2 =H R2 =H

2 3 6 8 10 11 14 16

R 1=S1 R 1=S1 R 1=S1 R 1=S2 R 1=S2 R 1=S2 R 1=S2 R 1=S2

R 2 =H R 2 =OH R 2 =H R 2 =OH R2 =H R2 =OH R2 =H R2 =OH

O OR 4

R2 H

OH

R 1O

OH HO HO

S1

O OH

H 3C

O

O

OH

OCH 3

OH

OH HO HO

R 3 =Ac R3 =Tig R 3 =Tig R 3 =Bz R 3 =Ac R 3 =Tig R 3 =Bz R 3 =Tig

R 4=Tig R4 =Tig R 4=Tig R 4=Tig R 4=Tig R 4=Tig R4 =Tig R 4=Bz

O 1 2

Ac

O

6

H R 3O

S2

1 4 5 7 9 12 13 15

7

3 4

18

12

O 20

O HO

O OH

H3 C

O OH

H 3C O OCH 3

R3 =Ac R 3=Bz R3 =Tig R 3=Bz R3 =Ac R 3=Bz R3 =Bz R 3=Tig

O H3 CO

O OH

R 4=Tig R 4 =Tig R4 =Tig R 4 =Tig R 4 =Tig R4 =Bz R 4=Tig R4 =Tig

3 2 4 5

Tig O 3 2

4

5

H3 C

Fig. 1. Structures of 1–16.

1

O 7 6

O H 3CO

1

O

Bz

70

X. Pang et al. / Steroids 93 (2015) 68–76

(750 mg) was loaded on ODS C18 column (2 cm  30 cm) eluted with CH3OH–H2O (v/v, 64:33–70:30) to give Fr.6 12–16 (each 50 mL) and Fr.6 17 (MeOH elution). Fr.6 12–16 (239.5 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 69:31, flow rate 4.0 mL/min) to afford another mixture D (42.6 mg, tR 35.3 min), and Fr.6 17 (360 mg) was separated by semi-preparative HPLC with CH3OH–H2O (v/v, 70:30, flow rate 4.0 mL/min) to give mixture E (76.7 mg, tR 37.3 min) and mixture F (84.7 mg, tR 40.0 min). Combined mixture D was purified by semi-preparative HPLC with CH3CN–H2O (v/v, 40:60, flow rate 3.5 mL/min) to give compound 11 (15.2 mg, tR 42.1 min). Mixture E was purified by semi-preparative HPLC with CH3CN–H2O (v/v, 40:60, flow rate 4.0 mL/min) to give compound 13 (15.2 mg, tR 32.4 min). Mixture F was purified by semi-preparative HPLC with CH3CN–H2O (v/v, 40:60, flow rate 4.0 mL/min) to give compounds 14 (48.7 mg, tR 40.6 min) and 15 (38.1 mg, tR 38.1 min). 2.3.1. Compound 1 C48H76O19; white amorphous powder; ½a25 D = +26.6 (c = 0.0754, CH3OH). HRESIMS (negative): m/z 955.4913 [MH] (calcd. for C48H75O19, 955.4903). 1H NMR data (600 MHz, pyridine-d5): d 3.80 (1H, m, H-3), 5.56 (1H, t, J = 10.2 Hz, H-11), 5.17 (1H, d, J = 9.8 Hz, H-12), 3.14 (1H, dd, J = 8.8, 5.0 Hz, H-17), 1.32 (3H, s, 18-CH3), 0.96 (3H, s, 19-CH3), 2.16 (3H, s, 21-CH3), 1.92 (3H, s, Ac-H-2), 7.15 (1H, qq, J = 7.2, 1.4 Hz, Tig-H-3), 1.67 (3H, d, J = 7.2 Hz, Tig-H-4), 1.96 (3H, s, Tig-H-5), 4.77 (1H, dd, J = 9.7, 1.5 Hz, Ole-H-1), 1.74 (1H, m, Ole-H-2a), 2.40 (1H, m, Ole-H-2b), 3.59 (2H, overlap, Ole-H-3, 5), 3.57 (1H, m, Ole-H-4), 1.64 (3H, d, J = 5.6 Hz, Ole-H-6), 3.50 (3H, s, Ole-3-OCH3), 5.27 (1H, d, J = 8.1 Hz, Allo-H-1), 3.80 (1H, m, Allo-H-2), 4.47 (1H, t, J = 2.7 Hz, Allo-H-3), 3.73 (1H, dd, J = 9.6, 2.7 Hz, Allo-H-4), 4.26 (1H, m, Allo-H-5), 1.63 (3H, d, J = 6.3 Hz, Allo-H-6), 3.81 (3H, s, Allo-3OCH3), 4.97 (1H, d, J = 7.7 Hz, Glc-H-1), 4.02 (1H, dd, J = 8.4, 7.7 Hz, Glc-H-2), 4.26 (1H, m, Glc-H-3), 4.20 (1H, dd, J = 9.2, 8.9 Hz, Glc-H-4), 3.98 (1H, m, Glc-H-5), 4.36 (1H, dd, J = 11.6, 5.4 Hz, Glc-H-6a), 4.53 (1H, dd, J = 11.6, 2.5 Hz, Glc-H-6b). 13C NMR data see Tables 1 and 2.

2.3.2. Compound 2 C48H74O19; white amorphous powder; ½a25 D = +23.6 (c = 0.0785, CH3OH). HRESIMS (negative): m/z 953.4778 [MH] (calcd. for C48H73O19, 953.4746). 1H NMR data (600 MHz, pyridine-d5): d 3.76 (1H, m, H-3), 5.50 (1H, br d, J = 5.6 Hz, H-6), 5.72 (1H, t, J = 10.2 Hz, H-11), 5.24 (1H, d, J = 10.2 Hz, H-12), 3.15 (1H, dd, J = 9.1, 4.8 Hz, H-17), 1.36 (3H, s, 18-CH3), 1.23 (3H, s, 19-CH3), 2.17 (3H, s, 21-CH3), 1.93 (3H, s, Ac-H-2), 7.15 (1H, q, J = 7.1 Hz, Tig-H-3), 1.68 (3H, d, J = 7.1 Hz, Tig-H-4), 1.97 (3H, s, Tig-H-5), 4.72 (1H, br d, J = 9.5 Hz, Ole-H-1), 3.50 (3H, s, Ole-3-OCH3), 5.26 (1H, d, J = 8.0 Hz, Allo-H-1), 3.81 (3H, s, Allo-3-OCH3), 4.97 (1H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data see Tables 1 and 2.

2.3.3. Compound 3 C53H76O20; white amorphous powder; ½a25 D = +46.0 (c = 0.0362, CH3OH). HRESIMS (negative): m/z 1031.4900 [MH] (calcd. for C53H75O20, 1031.4852). 1H NMR data (600 MHz, pyridine-d5): d 3.83 (1H, m, H-3), 5.43 (1H, br d, J = 5.4 Hz, H-6),6.61 (1H, t, J = 10.7 Hz, H-11), 5.59 (1H, d, J = 10.2 Hz, H-12), 3.22 (1H, m, H17), 1.71 (3H, s, 18-CH3), 1.69 (3H, s, 19-CH3), 2.18 (3H, s, 21CH3), 8.18 (2H, dd, J = 7.5, 1.8 Hz, Bz-H-3, 7), 7.37 (2H, d, J = 7.5, 7.2 Hz, Bz-H-4, 6), 7.45 (1H, t, J = 7.2 Hz, Bz-H-5), 6.89 (1H, q, J = 7.1 Hz, Tig-H-3), 1.42 (3H, d, J = 7.1 Hz, Tig-H-4), 1.61 (3H, s, Tig-H-5), 4.72 (1H, dd, J = 9.6, 1.2 Hz, Ole-H-1), 3.45 (3H, s, Ole-3OCH3), 5.23 (1H, d, J = 8.1 Hz, Allo-H-1), 3.80 (3H, s, Allo-3-OCH3), 4.96 (1H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data see Tables 1 and 2.

2.3.4. Compound 4 C51H80O20; white amorphous powder; ½a25 D = +36.6 (c = 0.0654, CH3OH). HRESIMS (negative): m/z 1011.5192 [MH] (calcd. for C51H79O20, 1011.5165). 1H NMR data (600 MHz, pyridine-d5): d 3.90 (1H, m, H-3), 6.48 (1H, t, J = 10.7 Hz, H-11), 5.42 (1H, d, J = 10.1 Hz, H-12), 3.27 (1H, dd, J = 9.2, 5.7 Hz, H-17), 1.64 (3H, s, 18-CH3), 1.51 (3H, s, 19-CH3), 2.15 (3H, s, 21-CH3), 6.96 (1H, q, J = 6.8 Hz, Tig1-H-3), 1.61 (3H, d, J = 7.1 Hz, Tig1-H-4), 1.81 (3H, s, Tig1-H-5), 7.05 (1H, q, J = 7.1 Hz, Tig2-H-3), 1.62 (3H, d, J = 7.1 Hz, Tig2-H-4), 1.88 (3H, s, Tig2-H-5), 4.82 (1H, br d, J = 9.7 Hz, Ole-H1), 3.50 (3H, s, Ole-3-OCH3), 5.29 (1H, d, J = 8.1 Hz, Allo-H-1), 3.83 (3H, s, Allo-3-OCH3), 4.99 (1H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data see Tables 1 and 2. 2.3.5. Compound 5 C51H80O19; white amorphous powder; ½a25 D = +27.6 (c = 0.0938, CH3OH). HRESIMS (negative): m/z 995.5262 [MH] (calcd. for C51H79O19, 995.5216). 1H NMR data (600 MHz, pyridine-d5): d 3.81 (1H, m, H-3), 5.73 (1H, t, J = 10.2 Hz, H-11), 5.24 (1H, d, J = 10.2 Hz, H-12), 3.17 (1H, dd, J = 9.3, 5.1 Hz, H-17), 1.36 (3H, s, 18-CH3), 1.03 (3H, s, 19-CH3), 2.16 (3H, s, 21-CH3), 6.91 (1H, q, J = 7.2 Hz, Tig1-H-3), 1.59 (3H, overlap, Tig1-H-4), 1.78 (3H, s, Tig1-H-5), 7.04 (1H, q, J = 7.1 Hz, Tig2-H-3), 1.63 (3H, d, J = 7.1 Hz, Tig2-H-4), 1.88 (3H, s, Tig2-H-5), 4.75 (1H, dd, J = 9.7, 1.5 Hz, OleH-1), 3.50 (3H, s, Ole-3-OCH3), 5.25 (1H, d, J = 8.0 Hz, Allo-H-1), 3.81 (3H, s, Allo-3-OCH3), 4.96 (1H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data see Tables 1 and 2. 2.3.6. Compound 6 C51H78O19; white amorphous powder; ½a25 D = +27.1 (c = 0.0685, CH3OH). HRESIMS (negative): m/z 993.5053 [MH] (calcd. for C51H77O19, 993.5059). 1H NMR data (600 MHz, pyridine-d5): d 3.77 (1H, m, H-3), 5.52 (1H, br d, J = 5.7 Hz, H-6), 5.88 (1H, t, J = 10.5 Hz, H-11), 5.30 (1H, d, J = 10.2 Hz, H-12), 3.18 (1H, dd, J = 9.3, 4.8 Hz, H-17), 1.38 (3H, s, 18-CH3), 1.31 (3H, s, 19-CH3), 2.17 (3H, s, 21-CH3), 6.92 (1H, q, J = 7.1 Hz, Tig1-H-3), 1.57 (3H, d, J = 7.1 Hz, Tig1-H-4), 1.78 (3H, s, Tig1-H-5), 7.06 (1H, q, J = 7.1 Hz, Tig2-H-3), 1.64 (3H, d, J = 7.1 Hz, Tig2-H-4), 1.89 (3H, s, Tig2-H-5), 4.73 (1H, br d, J = 9.5 Hz, Ole-H-1), 3.50 (3H, s, Ole-3-OCH3), 5.25 (1H, d, J = 8.0 Hz, Allo-H-1), 3.81 (3H, s, Allo-3-OCH3), 4.96 (1H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data see Tables 1 and 2. 2.3.7. Compound 7 C53H78O19; white amorphous powder; ½a25 D = +33.3 (c = 0.0631, CH3OH). HRESIMS (negative): m/z 1017.5092 [MH] (calcd. for C53H77O19, 1017.5059). 1H NMR data (600 MHz, pyridine-d5): d 3.79 (1H, m, H-3), 5.92 (1H, t, J = 10.4 Hz, H-11), 5.38 (1H, d, J = 10.3 Hz, H-12), 3.28 (1H, dd, J = 9.2, 5.1 Hz, H-17), 1.42 (3H, s, 18-CH3), 1.09 (3H, s, 19-CH3), 2.18 (3H, s, 21-CH3), 8.16 (2H, dd, J = 7.7, 1.8 Hz, Bz-H-3, 7), 7.41 (2H, dd, J = 7.7, 7.4 Hz, Bz-H-4, 6), 7.49 (1H, t, J = 7.4 Hz, Bz-H-5), 6.90 (1H, q, J = 7.1 Hz, Tig-H-3), 1.46 (3H, d, J = 7.1 Hz, Tig-H-4), 1.64 (3H, s, Tig-H-5), 4.73 (1H, br d, J = 9.7 Hz, Ole-H-1), 3.49 (3H, s, Ole-3-OCH3), 5.26 (1H, d, J = 8.1 Hz, Allo-H-1), 3.82 (3H, s, Allo-3-OCH3), 4.98 (1H, d, J = 7.7 Hz, Glc-H-1). 13C NMR data see Tables 1 and 2. 2.3.8. Compound 8 C59H86O25; white amorphous powder; ½a25 D = +38.8 (c = 0.0700, CH3OH). HRESIMS (negative): m/z 1193.5404 [MH] (calcd. for C59H85O25, 1193.5380). 1H NMR data (600 MHz, pyridine-d5): d 3.82 (1H, m, H-3), 5.43 (1H, br d, J = 5.3 Hz, H-6), 6.61 (1H, t, J = 10.6 Hz, H-11), 5.58 (1H, d, J = 10.1 Hz, H-12), 3.22 (1H, dd, J = 9.1, 4.8 Hz, H-17), 1.69 (3H, s, 18-CH3), 1.71 (3H, s, 19-CH3), 2.18 (3H, s, 21-CH3), 8.18 (2H, dd, J = 8.4, 1.3 Hz, Bz-H-3, 7), 7.37 (2H, dd, J = 7.8, 7.4 Hz, Bz-H-4, 6), 7.44 (1H, t, J = 7.4 Hz, Bz-H-5), 6.88 (1H, q, J = 7.7 Hz, Tig-H-3), 1.42 (3H, d, J = 7.1 Hz, Tig-H-4),

71

X. Pang et al. / Steroids 93 (2015) 68–76 Table 1 C chemical shifts of aglycones of 1–16 (150 MHz for 1–12, and 16, and 125 MHz for 13–15, pyridine-d5).

13

Position

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 11-O1 2 3 4 5 6 7 12-O1 2 3 4 5 6 7

38.1 30.3 76.2 35.4 44.7 29.4 28.4 40.0 50.1 37.9 71.7 78.2 54.9 83.9 34.0 24.3 58.4 11.8 12.4 213.6 31.7 Ac 170.4 21.5

38.8 30.4 77.3 39.8 139.7 122.6 28.2 37.5 47.9 39.5 71.9 77.7 54.8 84.1 34.6 24.0 58.3 11.6 19.3 213.7 31.8 Ac 170.2 21.4

39.5 30.0 76.3 35.7 45.7 25.4 35.2 78.4 51.4 38.5 71.3 78.9 55.7 85.5 36.3 24.8 59.4 14.1 13.4 214.0 31.8 Tig1 167.0 129.3 138.4 14.3 12.1

38.3 30.3 76.1 35.4 44.7 29.4 28.4 40.2 50.3 38.0 71.7 78.2 54.9 84.0 34.0 24.3 58.4 11.9 12.3 213.6 31.7 Tig1 167.4 129.1 138.4 14.3 12.0

38.8 30.4 77.3 39.8 139.8 122.5 28.3 37.6 48.1 39.5 71.9 77.7 54.8 84.2 34.8 24.0 58.3 11.7 19.3 213.8 31.8 Tig1 167.1 129.0 138.5 14.3 12.0

38.3 30.3 76.1 35.4 44.7 29.4 28.4 40.3 50.3 38.0 71.7 79.1 55.0 84.1 34.0 24.4 58.4 11.9 12.4 213.4 31.7 Tig 167.3 128.9 138.6 14.1 11.7

40.4 30.1 77.8 39.8 139.7 118.9 35.7 76.1 49.3 39.3 71.6 78.4 55.7 85.6 36.8 24.4 59.3 13.6 18.2 213.4 31.4 Tig1 167.0 129.1 138.4 14.3 12.0

Tig2 167.9 128.5 138.7 14.3 12.1

Tig 167.9 128.6 139.0 14.4 12.2

Tig 167.9 128.5 139.1 14.4 12.2

Tig2 167.9 128.6 138.6 14.3 12.1

38.3 30.2 76.0 35.4 44.7 29.4 28.5 40.3 50.3 38.1 72.6 78.1 55.0 84.0 34.0 24.4 58.4 11.9 12.4 213.6 31.7 Bz 166.4 130.9 130.1 128.8 133.4 128.8 130.1 Tig 167.8 128.3 138.7 14.2 11.8

38.7 30.3 77.2 39.8 139.8 122.6 28.3 37.7 48.1 39.5 72.8 77.6 54.9 84.2 34.8 24.1 58.3 11.7 19.4 213.8 31.8 Bz 166.1 130.8 1301 128.8 133.5 128.8 130.1

Tig2 167.9 128.6 138.5 14.3 12.1

40.5 30.0 77.6 39.8 139.6 119.0 35.6 76.2 49.4 39.4 72.5 79.3 55.8 85.7 36.9 24.4 59.4 13.7 18.2 213.1 31.4 Bz1 166.0 130.5 129.9 128.6 133.2 128.6 129.9 Bz 166.9 130.0 130.0 128.7 133.5 128.7 130.0

38.3 30.3 76.1 35.4 44.7 29.4 28.4 40.2 50.3 38.0 71.7 78.2 54.9 84.0 34.0 24.3 58.4 11.9 12.3 213.6 31.7 Tig1 167.4 129.1 138.4 14.3 12.0

Tig2 168.0 128.7 138.4 14.3 12.1

40.5 30.0 77.7 39.8 139.7 119.0 35.7 76.2 49.4 39.4 72.6 78.3 55.7 85.7 36.9 24.4 59.4 13.7 18.2 213.3 31.4 Bz 165.9 130.9 130.1 128.8 133.4 128.8 130.1 Tig 167.9 128.3 138.7 14.2 11.8

38.8 30.4 77.3 39.8 139.7 122.6 28.2 37.5 47.9 39.5 71.9 77.7 54.8 84.1 34.6 24.0 58.3 11.6 19.3 213.7 31.8 Ac 170.2 21.4

Tig 167.9 128.5 139.1 14.4 12.2

38.3 30.2 76.0 35.4 44.7 29.4 28.5 40.3 50.3 38.1 72.6 78.1 55.0 84.0 34.0 24.4 58.4 11.9 12.4 213.6 31.7 Bz 166.4 130.9 130.1 128. 8 133.4 128.8 130.1 Tig 167.8 128.3 138.7 14.2 11.8

38.1 30.3 76.2 35.4 44.7 29.4 28.4 40.0 50.1 37.9 71.7 78.2 54.9 83.9 33.9 24.3 58.4 11.8 12.4 213.6 31.7 Ac 170.4 21.5

Tig 167.9 128.6 139.0 14.4 12.2

40.0 30.2 77.9 40.7 139.8 119.1 37.0 76.4 49.6 39.6 72.1 78.6 55.9 85.8 35.8 24.6 59.5 13.5 18.5 213.5 31.6 Bz 166.1 131.1 130.3 129.3 133.6 129.3 130.3 Tig 168.1 128.5 138.9 14.4 12.0

Bz 166.8 130.3 130.1 128.9 133.7 128.9 130.1

Tig2 168.0 128.6 138.6 14.3 12.1

167.8 128.3 138.8 14.2 11.9

Table 2 C chemical shifts of sugar moieties of 1–16 (150 MHz for 1–12, and 16, and 125 MHz for 13–15, pyridine-d5).

13

Position

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Ole-1 -2 -3 -4 -5 -6 3-OCH3 Allo-1 -2 -3 -4 -5 -6 3-OCH3 Glc1-1 -2 -3 -4 -5 -6 Glc2-1 -2 -3 -4 -5 -6

97.7 37.9 79.7 83.3 72.0 19.1 57.2 101.9 72.7 83.2 83.4 69.5 18.3 61.7 106.6 75.5 78.4 72.0 78.4 63.0

98.0 37.8 79.6 83.2 71.9 19.0 57.3 101.9 72.7 83.2 83.4 69.5 18.3 61.7 106.6 75.5 78.4 71.9 78.4 63.0

98.1 37.9 79.8 83.3 72.1 18.5 57.2 102.0 72.8 83.3 83.5 69.7 18.5 61.9 106.6 75.7 78.6 72.1 78.5 63.5

97.5 37.8 79.7 83.3 71.9 19.1 57.2 101.9 72.7 83.2 83.3 69.5 18.3 61.7 106.6 75.5 78.4 71.9 78.4 63.0

97.5 37.8 79.6 83.2 71.9 19.1 57.2 101.9 72.7 83.2 83.4 69.5 18.3 61.7 106.6 75.5 78.4 71.9 78.4 63.0

98.0 37.8 79.6 83.2 71.9 19.0 57.2 101.9 72.7 83.2 83.3 69.5 18.3 61.7 106.6 75.5 78.4 71.9 78.4 63.0

97.5 37.8 79.6 83.2 71.9 19.0 57.2 101.9 72.7 83.3 83.3 69.5 18.3 61.7 106.6 75.5 78.4 71.9 78.4 63.0

97.9 37.7 79.6 83.2 71.9 19.0 57.1 101.8 72.7 83.1 83.4 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.6 78.6 62.5

97.7 37.8 79.7 83.3 72.0 19.1 57.2 101.9 72.7 83.1 83.5 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.5 105.0 74.8 78.3 71.6 78.6 62.5

98.0 37.8 79.6 83.2 71.9 19.0 57.2 101.9 72.7 83.1 83.4 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.6 78.6 62.5

97.9 37.7 79.6 83.3 71.9 19.0 57.3 101.9 72.7 83.1 83.4 69.4 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.5 78.6 62.5

97.5 37.8 79.6 83.3 71.9 19.1 57.2 101.9 72.7 83.1 83.4 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.6 78.6 62.5

97.5 37.8 79.6 83.2 71.9 19.0 57.2 101.9 72.7 83.1 83.4 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.6 78.6 62.5

97.9 37.7 79.6 83.2 71.9 19.0 57.2 101.9 72.65 83.1 83.4 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.5 78.6 62.5

97.5 37.8 79.6 83.3 71.9 19.1 57.2 101.9 72.7 83.1 83.4 69.5 18.3 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.5 78.6 62.5

98.0 37.7 79.6 83.2 71.9 19.0 57.2 101.9 72.7 83.1 83.4 69.5 18.2 61.7 106.2 75.0 76.6 81.5 76.4 62.4 105.0 74.8 78.3 71.6 78.6 62.5

72

X. Pang et al. / Steroids 93 (2015) 68–76

1.61 (3H, s, Tig-H-5), 4.72 (1H, dd, J = 9.7, 1.3 Hz, Ole-H-1), 1.66 (1H, m, Ole-H-2a), 2.33 (1H, m, Ole-H-2b), 3.52 (2H, overlap, OleH-3,5), 3.51 (2H, overlap, Ole-H-4, 5), 1.55 (3H, d, J = 3.1 Hz, OleH-6), 3.45 (3H, s, Ole-3-OCH3), 5.23 (1H, d, J = 8.1 Hz, Allo-H-1), 3.79 (1H, m, Allo-H-2), 4.38 (1H, t, J = 2.6 Hz, Allo-H-3), 3.65 (1H, dd, J = 9.6, 2.4 Hz, Allo-H-4), 4.23 (1H, m, Allo-H-5), 1.60 (3H, d, J = 6.5 Hz, Allo-H-6), 3.78 (3H, s, Allo-3-OCH3), 4.89 (1H, d, J = 7.7 Hz, Glc1-H-1), 3.99 (1H, dd, J = 8.2, 7.7 Hz, Glc1-H-2), 4.25 (1H, m, Glc1-H-3), 4.28 (1H, m, Glc1-H-4), 3.92 (1H, m, Glc1-H-5), 4.43 (1H, dd, J = 11.7, 2.6 Hz, Glc1-H-6a), 4.49 (1H, dd, J = 11.7, 3.6 Hz, Glc1-H-6b), 5.19 (1H, d, J = 7.8 Hz, Glc2-H-1), 4.09 (1H, dd, J = 8.2, 7.8 Hz Glc2-H-2), 4.20 (1H, m, Glc2-H-3), 4.18 (1H, m, Glc2-H-4), 4.01 (1H, m, Glc2-H-5), 4.29 (1H, dd, J = 11.5, 5.9 Hz, Glc2-H-6a), 4.53 (1H, dd, J = 11.5, 2.1 Hz, Glc2-H-6b). 13C NMR data see Tables 1 and 2. 2.3.9. Compound 9 C54H86O24; white amorphous powder; ½a25 D = +24.0 (c = 0.0654, CH3OH). HRESIMS (negative): m/z 1117.5391 [MH] (calcd. for C54H85O24, 1117.5431). 1H NMR data (600 MHz, pyridine-d5): d 3.81 (1H, m, H-3), 5.56 (1H, t, J = 10.2 Hz, H-11), 5.17 (1H, d, J = 9.8 Hz, H-12), 3.14 (1H, dd, J = 8.8, 5.0 Hz, H-17), 1.33 (3H, s, 18-CH3), 0.96 (3H, s, 19-CH3), 2.16 (3H, s, 21-CH3), 1.92 (3H, s, Ac-H-2), 7.15 (1H, q, J = 7.2 Hz, Tig-H-3), 1.67 (3H, d, J = 7.2 Hz, Tig-H-4), 1.96 (3H, s, Tig-H-5), 4.77 (1H, br d, J = 9.4 Hz, Ole-H-1), 3.50 (3H, s, Ole-3-OCH3), 5.27 (1H, d, J = 8.0 Hz, Allo-H-1), 3.81 (3H, s, Allo-3-OCH3), 4.90 (1H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2. 2.3.10. Compound 10 C54H84O24; white amorphous powder; ½a25 D = +16.7 (c = 0.1038, CH3OH). HRESIMS (negative): m/z 1115.5323 [MH] (calcd. for C54H83O24, 1115.5272). 1H NMR data (600 MHz, pyridine-d5): d 3.77 (1H, m, H-3), 5.50 (1H, br d, J = 5.6 Hz, H-6), 5.72 (1H, t, J = 10.2 Hz, H-11), 5.24 (1H, d, J = 10.2 Hz, H-12), 3.15 (1H, dd, J = 9.1, 4.8 Hz, H-17), 1.36 (3H, s, 18-CH3), 1.23 (3H, s, 19-CH3), 2.17 (3H, s, 21-CH3), 1.93 (3H, s, Ac-H-2), 7.16 (1H, q, J = 7.1 Hz, Tig-H-3), 1.68 (3H, d, J = 7.2 Hz, Tig-H-4), 1.98 (3H, s, Tig-H-5), 4.75 (1H, br d, J = 9.2 Hz, Ole-H-1), 3.49 (3H, s, Ole-3-OCH3), 5.23 (1H, d, J = 8.1 Hz, Allo-H-1), 3.78 (1H, s, Allo-3-OCH3), 4.89 (1H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2. 2.3.11. Compound 11 C61H84O25; white amorphous powder; ½a25 D = +63.3 (c = 0.0808, CH3OH). HRESIMS (negative): m/z 1215.5281 [MH] (calcd. for C61H83O25: 1215.5223). 1H NMR data (600 MHz, pyridine-d5): d 3.85 (1H, m, H-3), 5.47 (1H, br d, J = 4.7 Hz, H-6), 6.75 (1H, t, J = 10.7 Hz, H-11), 5.79 (1H, d, J = 10.0 Hz, H-12), 3.31 (1H, m, H17), 1.80 (3H, s, 18-CH3), 1.74 (3H, s, 19-CH3), 2.18 (3H, s, 21CH3), 8.04 (2H, d, J = 7.2 Hz Bz1-H-3, 7), 7.20 (2H, overlap, Bz1-H4, 6), 7.20 (1H, overlap, Bz1-H-5), 8.12 (2H, d, J = 7.7 Hz, Bz2-H-3, 7), 7.27 (2H, dd, J = 7.7, 7.4 Hz, Bz2-H-4, 6), 7.39 (1H, t, J = 7.4 Hz, Bz2-H-5), 4.75 (1H, br d, J = 9.6 Hz, Ole-H-1), 3.47 (3H, s, Ole-3OCH3), 5.25 (1H, d, J = 8.2 Hz, Allo-H-1), 3.80 (3H, s, Allo-3-OCH3), 4.92 (1H, d, J = 7.7 Hz, Glc1-H-1), 5.21 (1H, d, J = 7.8 Hz, Glc2-H-1). 13 C NMR data see Tables 1 and 2. 2.3.12. Compound 12 C57H90O24; white amorphous powder; ½a25 D = +25.1 (c = 0.0962, CH3OH). HRESIMS (negative): m/z 1157.5778 [MH] (calcd. for C57H89O24, 1157.5744). 1H NMR data (600 MHz, pyridine-d5): d 3.81(1H, m, H-3), 5.73 (1H, t, J = 10.5 Hz, H-11), 5.24 (1H, d, J = 10.2 Hz, H-12), 3.17 (1H, dd, J = 9.3, 5.1 Hz, H-17), 1.36 (3H, s, 18-CH3), 1.03 (3H, s, 19-CH3), 2.16 (3H, s, 21-CH3), 6.91 (1H, q, J = 7.2 Hz, Tig1-H-3), 1.59 (3H, overlap, Tig1-H-4), 1.78 (3H, s,

Tig1-H-5), 7.04 (1H, q, J = 7.1 Hz, Tig2-H-3), 1.63 (3H, d, J = 7.1 Hz, Tig2-H-4), 1.88 (3H, s, Tig2-H-5), 4.75 (1H, dd, J = 9. 4, 1.5 Hz, OleH-1), 3.48 (3H, s, Ole-3-OCH3), 5.25 (1H, d, J = 8.2 Hz, Allo-H-1), 3.79 (3H, s, Allo-3-OCH3), 4.90 (1H, d, J = 7.8 Hz, Glc1-H-1), 5.19 (1H, d, J = 7.9 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2. 2.3.13. Compound 13 C59H88O24; white amorphous powder; ½a25 D = +34.1 (c = 0.0715, CH3OH). HRESIMS (negative): m/z 1179.5624 [MH] (calcd. for C59H87O24, 1179.5587). 1H NMR data (500 MHz, pyridine-d5): d 3.78 (1H, m, H-3), 5.92 (1H, t, J = 10.4 Hz, H-11), 5.38 (1H, d, J = 10.2 Hz, H-12), 3.28 (1H, dd, J = 9.2, 5.2 Hz,H-17), 1.42 (3H, s, 18-CH3), 1.09 (3H, s, 19-CH3), 2.18 (3H, s, 21-CH3), 8.16 (2H, d, J = 7.8 Hz, Bz-H-3, 7), 7.37 (2H, dd, J = 7. 8, 7.4 Hz, Bz-H-4, 6), 7.44 (1H, t, J = 7.4 Hz, Bz-H-5), 6.90 (1H, q, J = 7.0 Hz, Tig-H-3), 1.46 (3H, d, J = 7.0 Hz, Tig-H-4), 1.64 (3H, s, Tig-H-5), 4.73 (br d, J = 9.7 Hz, Ole-H-1), 3.49 (3H, s, Ole-3-OCH3), 5.26 (1H, d, J = 8.1 Hz, Allo-H-1), 3.80 (3H, s, Allo-3-OCH3), 4.91 (1H, d, J = 7.8 Hz, Glc1-H-1), 5.21 (1H, d, J = 7.9 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2. 2.3.14. Compound 14 C59H86O24; white amorphous powder; ½a25 D = +39.2 (c = 0.0700, CH3OH). HRESIMS (negative): m/z 1177.5447 [MH] (calcd. for C59H85O24, 1177.5431). 1H NMR data (500 MHz, pyridine-d5): d 3.75 (1H, m, H-3), 5.55 (1H, br d, J = 5.4 Hz, H-6), 6.08 (1H, t, J = 10.3 Hz, H-11), 5.44 (1H, d, J = 10.0 Hz, H-12), 3.28 (1H, dd, J = 9.2, 4.8 Hz, H-17), 1.45 (3H, s, 18-CH3), 1.37 (3H, s, 19-CH3), 2.19 (3H, s, 21-CH3), 8.16 (2H, d, J = 7.8 Hz, Bz-H-3, 7), 7.40 (2H, dd, J = 7.8, 7.4 Hz, Bz-H-4, 6), 7.48 (1H, t, J = 7.4 Hz, Bz-H-5), 6.92 (1H, q, J = 7.0 Hz, Tig-H-3), 1.48 (3H, d, J = 7.0 Hz, Tig-H-4), 1.66 (3H, s, Tig-H-5), 4.72 (1H, br d, J = 9.7 Hz, Ole-H-1), 3.48 (3H, s, Ole-3-OCH3), 5.25 (1H, d, J = 8.0 Hz, Allo-H-1), 3.80 (3H, s, Allo-3OCH3), 4.92 (1H, d, J = 7.8 Hz, Glc1-H-1), 5.21 (1H, d, J = 7.9 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2. 2.3.15. Compound 15 C59H88O24; white amorphous powder; ½a25 D = +29.1 (c = 0.0700, CH3OH). HRESIMS (negative): m/z 1179.5586 [MH] (calcd. for C59H87O24, 1179.5587). 1H NMR data (500 MHz, pyridine-d5): d 3.83 (1H, m, H-3), 5.87 (1H, t, J = 10.4 Hz, H-11), 5.43 (1H, d, J = 9.9 Hz, H-12), 3.28 (1H, dd, J = 8.7, 5.1 Hz, H-17), 1.46 (3H, s, 18-CH3), 1.07 (3H, s, 19-CH3), 2.08 (3H, s, 21-CH3), 6.78 (1H, q, J = 7.1 Hz, Tig-H-3), 1.40 (3H, d, J = 7.1 Hz, Tig-H-4), 1.54 (3H, s, Tig-H-5), 8.27 (2H, d, J = 7.7 Hz, Bz-H-3, 7), 7.45 (2H, dd, J = 7.7, 7.4 Hz, Bz-H-4, 6), 7.44 (1H, t, J = 7.4 Hz, Bz-H-5), 4.73 (1H, br d, J = 9.7 Hz, Ole-H-1), 3.49 (3H, s, Ole-3-OCH3), 5.26 (1H, d, J = 8.1 Hz, Allo-H-1), 3.80 (3H, s, Allo-3-OCH3), 4.91 (1H, d, J = 7.8 Hz, Glc1-H-1), 5.21 (1H, d, J = 7.9 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2. 2.3.16. Compound 16 C57H88O25; white amorphous powder; ½a25 D = +36.4 (c = 0.0531, CH3OH). HRESIMS (negative): m/z 1171.5635 [MH] (calcd. for C57H87O25, 1171.5689). 1H NMR data (600 MHz, pyridine-d5): d 3.84 (1H, m, H-3), 5.41 (1H, br d, J = 5.3 Hz, H-6), 6.43 (1H, t, J = 10.5 Hz, H-11), 5.46 (1H, d, J = 10.2 Hz, H-12), 3.19 (1H, dd, J = 9.1, 4.8 Hz,H-17), 1.67 (3H, s, 18-CH3), 1.65 (3H, s, 19-CH3), 2.17 (3H, s, 21-CH3), 6.93 (1H, q, J = 7.1 Hz, Tig1-H-3), 1.58 (3H, d, J = 7.1 Hz, Tig1-H-4), 1.80 (3H, s, Tig1-H-5), 7.03 (1H, q, J = 7.1 Hz, Tig2-H-3), 1.58 (3H, d, J = 7.1 Hz, Tig2-H-4), 1.88 (3H, s, Tig2-H-5), 4.75 (1H, dd, J = 9.7, 1.3 Hz, Ole-H-1), 3.46 (3H, s, Ole-3-OCH3), 5.24 (1H, d, J = 8.1 Hz, Allo-H-1), 3.78 (3H, s, Allo-3-OCH3), 4.90 (1H, d, J = 7.7 Hz, Glc1-H-1), 5.19 (1H, d, J = 7.8 Hz, Glc2-H-1). 13C NMR data see Tables 1 and 2.

73

X. Pang et al. / Steroids 93 (2015) 68–76

3. Results and discussion

H

The 95% EtOH extract from the roots of M. tenacissima was partitioned between EtOAc and H2O. The EtOAc extract was fractionated on silica gel column chromatography and further separated chromatographically on an MCI gel column. Finally, by repeated semi-preparative HPLC with different elution, sixteen new compounds were obtained. All the compounds were white amorphous powders and positive to Liebermann-Burchard and Keller-Kiliani reactions, suggesting they were steroidal glycosides with 2-deoxysugar moieties. Based on the spectrographic analyses of HRESIMS and NMR, compounds 1–16 exhibited the structural patterns of C21-steroid diester derivatives with the oligosaccharide sugar moiety consisting three or four units. 3.1. Compound 1 The HRESIMS showed a [MH] ion at m/z 955.4913, from which we deduced the molecular formula of compound 1 to be C48H76O19. The 1H NMR spectrum showed the methyl singlets at d 1.32 (3H, s, CH3-18), 0.96 (3H, s, CH3-19), and 2.16 (3H, s, CH321), and three methine protons indicative of secondary alcoholic functions at d 3.80 (1H, m, H-3), 5.56 (1H, t, J = 10.2 Hz, H-11), and 5.17 (1H, d, J = 9.8 Hz, H-12). Combination of the 1H and 13C NMR data indicated a C21 steroidal skeleton for the aglycone moiety of 1. The proton signals of d 1.92 (3H, s, Ac-H-2), 7.15 (1H, qq, J = 7.2, 1.4 Hz, Tig-H-3), 1.67 (3H, d, J = 7.2, Tig-H-4), and 1.96 (3H, s, Tig-H-5) and the carbon signals of d 170.4 (Ac-C-1), 21.5 (Ac-C-2) and 167.9 (Tig-C-1), d 128.6 (Tig-C-2), 139.0 (Tig-C-3), 14.4 (Tig-C4), and 12.2 (Tig-C-5) indicated the existence of an acetyl group and a tigloyl group in the molecule. The positions of the acetyl and tigloyl groups were determined to be located at C-11 and C12, respectively, based on the long-range correlations between d 170.4 (Ac-C-1) and 5.56 (H-11), and between d 167.9 (Tig-C-1) and 5.17 (H-12) in the HMBC spectrum (Fig. 2). The partial structure of C-9  C-12 were confirmed by 1H–1H COSY correlations between d 1.46 (H-9) and 5.56 (H-11), and between d 5.56 (H11) and 5.17 (H-12). By comparing the NMR data of the C21 steroidal skeleton of 1 with those of condurangosides A and B, we could identify the C21 steroid skeleton as (5a,8b,9a,17a)-20-one3b,11a,12b,14b-tetradroxypregnane (deacylcondurangogenin A) [14]. The relative configurations of the aglycone were further confirmed by correlations of H-3/H-5 in ROESY spectrum (Fig. 3). Thus, the aglycone structure of 1 was identified as 11-O-acetyl-12-O-tigloyl-deacylcondurangogenin A. By analyses of 1H–1H COSY, HSQC and HMBC spectra, all the proton and carbon signals of aglycone were assigned. The anomeric regions in the 1H and 13C NMR spectra of 1 showed three anomeric protons at d 4.77 (1H, dd, J = 9.7, 1.5 Hz), 5.27 (1H, d, J = 8.1 Hz), and 4.97 (1H, d, J = 7.7 Hz) and three anomeric carbon signal at d 97.7, 101.9, and 106.6, suggesting three sugar units existed in 1. Two methyl doublets at d 1.64

O

HMBC (Hൺ C) 1H-1H

O

O O

COSY

OH

OH HO HO

O

H 3C

O

O

OH

OCH3

O OH

H3C O H 3CO

O

O

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

H

H

H R 1O

H

H

H

H

OR3

CH3 R2 O

O

HO H

H H

H 3C

CH3

H

H

H

H

H H H

H

Fig. 3. Key ROESY correlations of the aglycone for 1.

(3H, d, J = 5.6 Hz) and 1.63 (3H, d, J = 6.3 Hz) and two methyl singlets at d 3.50 (3H, s) and 3.81 (3H, s) in the 1H NMR spectrum indicated the presence of two 6-deoxy-3-O-methyl pyranoses [15]. The sugar units were identified as oleandrose, 6-deoxy-3-Omethyl-allose and glucose by NMR spectroscopic data analysis, as well as by comparison with previously reported values finally. By using 1H–1H COSY, HSQC and HMBC spectra, we fully assigned the proton spin systems and the carbon resonances of each sugar. The large coupling constants (3J1,2 > 7 Hz) were consistent with the b configurations of the sugars. The connectivity of the sugars was established by the HMBC correlations between d 5.27 (Allo-H-1) and 83.3 (Ole-C-4), and d 4.97 (Glc-H-1) and 83.4 (Allo-C-4) (Fig. 2). Therefore, the sugar moiety was deduced as 3-O-b-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-allopyranosyl-(1?4)-boleandropyranoside which coincided with neo-condurangotriose in the compounds isolated from the same plant [5]. Furthermore, the glycosidation site was confirmed by HMBC correlation between d 4.77 (Ole-H-1) and 76.2 (C-3). Thus, the structure of 1 was determined to be 3-O-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methylb-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a-O-acetyl-12bO-tigloyl-deacylcondurangogenin A, named marstenacisside A1. 3.2. Compound 2 Compound 2 was suggested to had a molecular formula of C48H74O19 determined by HRESIMS [MH] ion at m/z 953.4778, two mass units less than that of 1. Comparison of the NMR data of 2 with those of 1 revealed that they had same sugar moiety, diester groups and almost identical C21 steroid skeleton except the differences in the A ring. A double bond at C-5 and C-6 could be deduced from the chemical shift of C-3 to C-6 (d 77.3, 39.8, 139.7, and 122.6) and H-6 (d 5.50, 1H, br d, J = 5.6). That was also confirmed by the 1H–1H COSY correction of H-6/H-7, and HMBC correlations of H-6/C-10, H-6/C-7, and H-6/C-8. Thus, the C21 steroid skeleton of 2 was deduced as (8b,9a,17a)-5-en-20-one3b,11a,12b, 14b-tetradroxypregnane which agreed with drevogenin P [16]. The whole structure of 2 was further confirmed by the combined use of 1H–1H COSY, HSQC, and HMBC experiments. Therefore, the structure of 2 was concluded to be 3-O-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)b-D-oleandropyranosyl-11a-O-acetyl-12b-O-tigloyl-drevogenin P, named marstenacisside A2. 3.3. Compound 3 Compound 3 had a molecular formula of C53H76O20 determined by HRESIMS ion [MH] at m/z 1031.4900. The NMR data of 3 suggested it contained the same sugar moiety as 2 with a different C21 steroid skeleton and diester groups. Comparison of the 13C NMR data of C21 steroid skeleton between 3 and 2 revealed significant chemical shift differences. The significant chemical shifts at C-7 (d 37.1, +8.9 ppm) and C-8 (d 76.4, +38.9 ppm) of 3 suggested it had one more hydroxyl group at C-8 than 2. Thus, by NMR

74

X. Pang et al. / Steroids 93 (2015) 68–76

spectroscopic data analysis as well as by comparison with previously reported data, the C21 steroid skeleton of 3 was deduced as (9a,17a)-5-ene-20-one-3b,8b,11a,12b,14b-pentahydroxypregnane coincided with marsdenin [14,17]. The proton signals of 8.18 (2H, dd, J = 7.5, 1.8 Hz, Bz-H-3, 7), 7.37 (2H, dd, J = 7.5, 7.2 Hz, Bz-H-4, 6), d 7.45 (1H, d, J = 7.2, Bz-H-5), 6.89 (1H, qq, J = 7.1, 1.5 Hz, TigH-3), 1.42 (3H, d, J = 7.1 Hz, Tig-H-4), and 1.61 (3H, s, Tig-H-5) together with the carbon signals of 166.1 (Bz-C-1), 131.1 (Bz-C2), 130.3 (Bz-C-3, 7), 129.0 (Bz-C-4, 6), 133.6 (Bz-C-5), 168.1 (TigC-1), d 128.5 (Tig-C-2), 138.9 (Tig-C-3), 14.4 (Tig-C-4), and 12.0 (Tig-C-5) deduced the existence of a benzoyl group and a tigloyl group in 3. The HMBC correlations between d 166.1 (Bz-C-1) and 6.61 (H-11), and between d 167.9 (Tig-C-1) and 5.59 (H-12) determined the linkages of the benzoyl group to C-11 and the tigloyl group to C-12. Finally, the structure of 3 was elucidated as 3-Ob-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl(1?4)-b-D-oleandropyranosyl-11a-O-benzoyl-12b-O-tigloyl-marsdenin, named marstenacisside A3. 3.4. Compound 4 The molecular formula of 4 was determined to be C51H80O20 by HRESIMS ion [MH] at m/z 1011.5192. By comparing the NMR data of 4 with those of 1, we deduced that 4 contained the same sugar chain and almost identical C21 steroid skeleton as 1. The significant 13C NMR shift differences of C-7 (d 35.2, +6.8 ppm) and C-8 (d 78.4, +38.4 ppm) suggested 4 had one more hydroxyl group at C8 than 1. Subsequently, by NMR spectroscopic data analysis and by comparison with previously reported data, the C21 steroid skeleton of 4 was deduced as (9a,17a)-5-ene-20-one-3b,8b,11a,12b,14bpentahydroxypregnane well coincided with tenacigenin C [5,7,14]. In the 13C NMR spectrum of 4, the carbon signals at d 168.1, 167.0, 138.4, 138.4, 129.3, 128.5, 14.4, 14.3, 12.1, and 12.1 suggested two tigloyl groups existed in the molecule. The structure of 4 was confirmed by the combined use of 1H–1H COSY, HSQC and HMBC experiments. Thus, 4 was identified to be 3-O-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-Doleandropyranosyl-11a, 12b-O-ditigloyl-tenacigenin C, named marstenacisside A4. 3.5. Compound 5 Compound 5, with the molecular formula of C51H80O19 determined by HRESIMS ion [MH] at m/z 995.5262, had almost the same structure as 1 except for the diester groups, based on comparison of their NMR data. In the 13C NMR spectrum, two groups of carbon signals at d 167.9, 167.4, 138.5, 138.4, 129.1, 128.6, 14.3, 14.3, 12.1, and 12.0 suggested two tigloyl groups existed in 5. Combined analysis of 1H–1H COSY, HSQC and HMBC spectra further confirmed the structure of 5. Accordingly, the structure of 5 was identified to be 3-O-b-D-glucopyranosyl-(1?4)-6-deoxy3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a, 12b-O-ditigloyl-deacylcondurangogenin A, named marstenacisside A5. 3.6. Compound 6 Compound 6 had the molecular formula of C51H78O19 determined by HRESIMS ion [MH] at m/z 993.5053. Comparison of the NMR data suggested that 6 had the same C21 steroid skeleton and sugar moieties as 2 and the same ditigloyl groups as 5. Therefore, the structure of 6 was identified to be 3-O-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-Doleandropyranoside-11a, 12b-O-ditigloyl-drevogenin P, named marstenacisside A6.

3.7. Compound 7 Compound 7 was established to have a molecular formula of C53H78O19 by the [MH] ion at m/z 1017.5092 in the HRESIMS. A detailed comparison of the NMR data of 7 with those of 1 and 3 revealed that 7 shared the same sugar moiety and C21 steroid skeleton with 1 and shared the same diester groups with 3. The structure of 7 was finally confirmed by combination of 1H–1H COSY, HSQC and HMBC experiments. Thus, 7 was identified as 3-O-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranoside-11a-O-benzoyl-12b-O-tigloyl-deacylcondurangogenin A, named marstenacisside A7. 3.8. Compound 8 Compound 8, with a molecular formula of C59H86O25 determined by HRESIMS ion [MH] at m/z 1193.5404, had 162 Da more than that of 3. The NMR data suggested 8 had the almost identical structure with 3 except the difference of one more terminal sugar in the sugar moiety of 8. In the anomeric regions of 1H and 13C NMR spectra of 8, four anomeric protons at d 5.23 (1H, d, J = 8.1 Hz), 5.19 (1H, d, J = 7.8 Hz),4.89 (1H, d, J = 7.7 Hz), and 4.72 (1H, dd, J = 9.7, 1.3 Hz), and four anomeric carbon signals at d 106.2, 105.0, 101.8, and 97.9 were observed. By analysis of NMR spectroscopic data as well as by comparison with previously reported values, it was deduced that the sugar moiety was comprised of an oleandrose, a 6-deoxy-3-O-methyl-allose and two glucoses. Using 1H–1H COSY, HSQC and HMBC experiments, we were able to fully assign the proton spin systems and the carbon resonances of each sugar. The b configurations of the sugars were determined by the large coupling constants (3J1,2 > 7 Hz) of each sugar. The HMBC correlations between d 4.72 (Ole-H-1) and 77.7 (C-3), between d 5.23 (Allo-H-1) and 83.2 (Ole-C-4), between d 4.89 (Glc1-H-1) and 83.4 (Allo-C-4), and between d 5.19 (Glc2-H1) and d 81.5 (Glc1-C-4) deduced the glycosidation site and the sequence of the sugar chain. Therefore, the sugar moiety was deduced as 3-O-b-glucopyranosyl-(1?4)-b-glucopyranosyl-(1?4)6-deoxy-3-O-methyl-b-allopyranosyl-(1?4)-b-oleandropyranoside that agreed well with those of other constituents of the same plant [8–9,11]. Therefore, 8 was elucidated as 3-O-b-D-glucopyranosyl(1?4)-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a-O-benzoyl-12b-Otigloyl-marsdenin, named marstenacisside B1. 3.9. Compound 9 Compound 9 had a molecular formula of C54H86O24 determined by HRESIMS ion [MH] at m/z 1117.5391, 162 Da more than that of 1. A detailed comparison of the NMR data of 9 with those of 1 and 8 revealed that 9 and 1 shared the same aglycone and diester groups, and 9 and 8 shared the same sugar moiety. Thus, the structure of 9 was deduced as 3-O-b-D-glucopyranosyl-(1?4)b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a-O-acetyl-12b-O-ditigloyldeacylcondurangogenin A, named marstenacisside B2. 3.10. Compound 10 The molecular formula of 10 was determined to be C54H84O24 based on the [MH] ion at m/z 1115.5323 in its HRESIMS spectrum. Comparison of the NMR and MS data of 10 with those of 2 and 8 indicated that 10 had the same aglycone as 2 and the same sugar moiety as 8. Consequently, the structure of 10 was assigned as 3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranosyl-(1?4)-6deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl11a-O-acetyl-12b-O-tigloyl-drevogenin P, named marstenacisside B3.

X. Pang et al. / Steroids 93 (2015) 68–76

3.11. Compound 11 Compound 11 had a molecular formula of C61H84O25 determined by HRESIMS ion [MH] at m/z 1215.5281. The NMR data suggested that 11 had a structure almost identical with that of 8, only with differences in the ester group at C-12. The lack of one tigloyl group but the presence of one benzoyl group at C-12 in 11 was deduced from the carbon signals at d 166.9, 133.5, 130.0 (3), and 128.7 (2) instead of d 167.9, 128.3, 138.7, 14.2, and 11.8 observed in the 13C NMR spectrum. Consequently, the structure of 11 was elucidated as 3-O-b-D-glucopyranosyl-(1?4)b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl(1?4)-b-D-oleandropyranosyl-11a,12b-O-dibenzoyl-marsdenin, named marstenacisside B4. 3.12. Compound 12 Compound 12 showed an [MH] ion peak at m/z 1157.5778 in the negative HRESIMS corresponding to a molecular formula of C57H90O24. By comparing the NMR and MS data of 12 with those of 5 and 8, we determined that 12 had the same aglycone as 5 and the same sugar moiety as 8. Thus, the structure of 12 was deduced as 3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranosyl(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a,12b-O-ditigloyl-deacylcondurangogenin A, named marstenacisside B5. 3.13. Compound 13 Compound 13, with a molecular formula of C59H88O24 determined by HRESIMS ion [MH] at m/z 1179.5624, had the same aglycone structure as 7 based on comparison of their NMR data. Moreover, the NMR data of 13 also suggested it had the same sugar moiety as 8. Thus, the structure of 13 was deduced as 3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methylb-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a-O-benzoyl-12 b-O-tigloyl-deacylcondurangogenin A, named marstenacisside B6. 3.14. Compound 14 The molecular formula of compound 14 was C59H86O24 as determined by HRESIMS ion [MH] at m/z 1179.5627 ie two Da less than that of 13. Comparison of NMR between 14 and 13 revealed they had the same structure except for the A ring. Further comparison of NMR data between 14 and 2 suggested they had the same C21 steroid skeleton. Thus, the structure of 14 was deduced as 3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranosyl-(1?4)-6-deoxy3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11 a-O-benzoyl-12b-O-tigloyl-drevogenin P, named marstenacisside B7. 3.15. Compound 15 Compound 15, with a molecular formula of C59H88O24 (HRESIMS ion [MH] at m/z 1179.5586), was identified as a isomer of 13. The 13C NMR data of 15 suggested it had the almost identical carbon signals with 13. However, the carbon chemical shifts of 15 at d 71.68 (C-11) and 79.13 (C-12), obviously different with those of 13 at d 72.6 (C-11) and 78.1 (C-12), suggested that 15 had the same diester groups as 13, but with different esterification positions. In the HMBC spectrum, the correlations between d 5.87 (H-11) and 167.34 (C-1 of Tig), and between d 5.43 (H-12) and 166.8 (C-1 of Bz) determined the positions of the tigloyl group at C-11 and benzoyl group at C-12, respectively. Thus, the

75

structure of 15 was deduced as 3-O-b-D-glucopyranosyl-(1?4)b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl(1?4)-b-D-oleandropyranosyl-11a-O-tigloyl-12b-O-benzoyl-deacylcondurangogenin, named marstenacisside B8. 3.16. Compound 16 Compound 16 had a molecular formula of C57H88O25 (HRESIMS ion [MH] at m/z 1171.5635). The 13C NMR data of 16 suggested it had the same sugar moiety and C21 steroid skeleton as 3, but with different diester groups. In the 13C NMR spectrum of 16, the carbon signals at d 168.0, 167.0, 138.6, 138.4, 129.2, 128.6, 14.3, 14.3, 12.1, and 12.0, like those of 4, suggested two tigloyl groups existed in the molecule. Thus, the structure of 16 was deduced as 3-O-b- D -glucopyranosyl-(1?4)-b- D -glucopyranosyl-(1?4)-6deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-oleandropyranosyl-11a,12b-O-ditigloyl-marsdenin, named marstenacisside B9. 4. Conclusion This paper presented the isolation and structure elucidation of sixteen new polyoxypregnane glycosides from the roots of M. tenacissima. The patterns of sugar moiety, C21 steroid skeleton, and the ester groups of these compounds were in agreement with those of compounds preciously isolated from the stems of this plant. This is the first time for systematic study on the polyoxypregnane glycosides from the roots of M. tenacissima. This work not only could be helpful to understand the constituents of the Dai medicine ‘dai-bai-jie’, but also could be helpful to understand the constituents in the different part of M. tenacissima, benefiting the investigation on the bioactive compounds from this plant. Conflict of interest There are no conflicts of interests of all authors. Acknowledgement Thanks to Miss. Meifeng Xu in National Center of Biomedical Analysis (NCBA) for the measurements of the NMR spectra. Appendix A. Supplementary data HRESIMS and NMR spectra of compounds 1–16 are available as supporting information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.steroids.2014.11.004. References [1] Li HT, Kang LP, Guo BL, Zhang ZL, Guan YH, Pang X, et al. Original plant identification of Dai nationality herb ‘‘daibaijie’’. Chin J Chin Mater Med 2014;39:27–31. [2] Qiu SX, Luo SQ, Lin LZ, Cordell GA. Further polyoxypregnane glycosides from Marsdenia tenacissima. Phytochemistry 1996;41:1385–8. [3] Xia ZH, Xing WX, Mao SL, Lao AN, Uzawa J, Yoshida S, et al. Pregnane glycosides from the stems of Marsdenia tenacissima. J Asia Nat Prod Res 2004;6:79–85. [4] Deng J, Liao ZX, Chen DF. Marsdenosides A–H Polyoxypregnane glycosides from Marsdenia tenacissima. Phytochemistry 2005;66:1040–51. [5] Deng J, Liao ZX, Chen DF. Two new C21 steroids from Marsdenia tenacissima. Chin Chem Lett 2005;16:487–90. [6] Wang S, Lai YH, Tian B, Yang L. Two new C21 steroidal glycosides from Marsdenia tenacissima (ROXB.)WIGHT et ARN. Chem Pharm Bull 2006;54: 696–8. [7] 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;89:2738–44. [8] Li QF, Wang XL, Ding LS, Zhang C. Polyoxypregnanes from the stems of Marsdenia tenacissima. Chin Chem Lett 2007;18:831–4. [9] Liu J, Yu ZB, Ye YH, Zhou YW. A new C21 steroid glycoside from Marsdenia tenacissima. Chin Chem Lett 2008;19:444–6.

76

X. Pang et al. / Steroids 93 (2015) 68–76

[10] Huang XD, Liu T, Wang S. Two new polyoxypregnane glycosides from Marsdenia tenacissima. Helv Chim Acta 2009;92:2111–7. [11] Wang XL, Peng SL, Ding LS. Further polyoxypregnane glycosides from Marsdenia tenacissima. J Asia Nat Prod Res 2010;12:654–61. [12] Zhang H, Tan AM, Zhang AY, Chen R, Yang SB, Huang X. Five new C21 steroidal glycosides from the stems of Marsdenia tenacissima. Steroids 2010;75: 176–83. [13] Xia ZH, Mao SL, Lao AN, Uzawa Jun, Yoshida S, Fujimoto Y. Five new pregnane glycosides from the stems of Marsdenia tenacissima. J Asia Nat Prod Res 2011;13:477–85.

[14] Umehara K, Endoh M, Miyase T, Kuroyanagi M, Ueno A. Studies on differentiation inducers. IV. Pregnane derivatives from condurango cortex. Chem Pharm Bull 1994;42:611–6. [15] Ma BX, Fang TZ. Novel saponins hainaneosides A and B isolated from Marsdenia hainanensis. J Nat Prod 1997;60:134–8. [16] Niranjan PS, Nilendu P, Nirup BM, Sukdeb B, Kazuo K, Tamotsu N. Polyoxypregnane glycosides from the flowers of Dregea volubilis. Phytochemistry 2002;61:383–8. [17] Warashina T, Noro T. Cardenolide and oxypregnane glycosides from the root of Asclepias incarnata L. Chem Pharm Bull 2000;48:516–24.