Steroidal Saponins from Myriopteron extensum and Their Cytotoxic Activity

• Abstract
• Introduction
• Materials and Methods
• General experimental procedure
• Plant material
• Extraction and isolation
• Cytotoxicity assays
• Results and Discussion
• Acknowledgements
• References

Abstract

Two new steroidal saponins, extensumsides A (1) and B (2), were isolated from the whole plants of Myriopteron extensum (Wight) K. Schum. Their structures were elucidated as 17β-uzarigenin-3-O-β-glucopyranosyl-(1→6)-β-glucopyranosyl-(1→4)-β-thevetopyranosyl-(1→4)-β-cymaropyranoside (1) and 12-(3-methylbut-2-enoyloxy)pregn-5-en-20-one 3-O-[β-cymaropyranosyl-(1→4)-β-cymaropyranosyl-(1→4)-β-thevetopyranosyl-(1→4)-(6-O-sulfo-β-glucopyranoside)] (2) on the basis of chemical and spectral evidence. Extensumside A exhibited significant cytotoxicity against eight cancer cell lines with a mean GI₅₀ value of 0.346 μg/mL.

Introduction

Myriopteron extensum (Wight) K. Schum, a unique species of the genus Myriopteron (Asclepiadaceae), is found in China, Vietnam, Laos, Burma, India and Thailand [1]. This plant is adapted to phthisis and the water extract of its roots is used in folk medicine as a remedy for cough at Yuanjiang, Yunnan Province of China [2]. However, no chemical study on this plant was reported.

Cardenolides show an inotropic action mainly due to the inhibition of Na⁺,K⁺-ATPase (NKA), which plays a crucial role in cellular ion homeostasis [3] and in membrane transport of selected cellular proteins and cationic substances important to tumor cell growth [4]. The family Asclepiadaceae is a rich source of cardenolides. In a preliminary screening, the 90 % EtOH extract of M. extensum displayed cytotoxicity. After fractionation of this extract in water by EtOAc and n-BuOH, the n-BuOH fraction showed the stronger cytotoxic activity. The separation of the n-BuOH fraction provided two new steroidal saponins, extensumsides A (1) and B (2) (Fig. [1]). In this paper, we report their structure elucidation and cytotoxic activities.

Materials and Methods

General experimental procedure

Melting points were determined on an XRC-1 micro-melting point apparatus and are uncorrected. Optical rotation was measured with a Perkin-Elmer 241 automatic polarimeter. IR (KBr discs) and UV (methanol as solvent) spectra were recorded on Nicolet Protege 460 and Lambda 35 spectrometers, respectively. Mass spectra were obtained on a Finnigan LCQ^DECA ion trap mass spectrometer (ESI), a HP110-LC/MS spectrometer (APCI), and a VG AutoSpec-3000 spectrometer (HRFAB). NMR spectra were recorded in C₅D₅N on Bruker AMX 500 and Advance 600 spectrometers, with tetramethylsilane (TMS) as internal reference, coupling constants were reported in Hz. Silica gel H, Sephadex LH-20, Macroporous resin D101 (pore size 13 - 14 nm, 26 - 60 mesh), and RP-C18 (40 - 63 μm) were used for column chromatography.

Plant material

The whole plants of M. extensum (Wight) K. Schum were collected in August 2000 from Xishuangbanna, Yunnan Province of China, and identified by Prof. Jing-Yun Cui in Xishuangbanna Tropical Botanical Garden, the Chinese Academy of Sciences. A voucher specimen (GF-123) is deposited at the Herbarium of Chengdu Institute of Biology, the Chinese Academy of Sciences.

Extraction and isolation

The air-dried whole plants of M. extensum (Wight) K. Schum (5 kg) were powdered and soaked with 90 % EtOH at room temperature (20 L × 7 days × 3). After concentrating under vacuum, ca. 305 g residue were obtained. A suspension of the residue in water (1 L) was partitioned with ethyl acetate (1 L × 10) and n-butanol (1 L × 10) to give 78 g and 142 g extract, respectively. The n-butanol fraction showed reasonable cytotoxicity (Table [2]) and was further chromatographed over a macroporous resin D101 column (13 × 62 cm, 2 kg), eluted first with water (10 L), and then with 50 % (5.5 L) and 85 % EtOH (5 L) to give fractions A (15.35 g) and B (45.35 g). Fraction A was subjected to silica column chromatography (8 × 60 cm, 160 - 200 mesh, 750 g), which was eluted with CHCl₃/CH₃OH/H₂O (5 : 1 : 0.01). The eluate of 900 to 1000 mL (560 mg) was submitted to RP-C18 column chromatography (3 × 30 cm, 80 g) eluted with CH₃OH/H₂O (3 : 2) to afford 1 (37 mg, 100 to 350 mL). Fraction B was subjected to a Sephadex LH-20 column (8 × 60 cm, 500 g), eluted with water to give the crude saponins (20.02 g). Part of this fraction (1.89 g) was separated on silica column chromatography (5 × 40 cm, 200 - 300 mesh, 160 g) with the solvents CHCl₃/CH₃OH/H₂O (4 : 1:0.01) to yield 2 (22 mg, 500 to 800 mL).

Extensumside A (1): White amorphous powder, C₄₉H₇₈O₂₁; m. p. 176 - 178 °C; [α]_D ²⁵: -12.5 (c 0.4, CH₃OH); IR (KBr): ν _max = 3426, 3402, 3339, 2935, 1737, 1632, 1451, 1379, 1168, 1078 cm^-1; UV (MeOH): λ_max (log ε) = 196 (0.67), 209 (1.12), 221 (2.38) nm. HRFABMS: m/z = 1001.4986 [M - H]^- (calcd.: 1001.4957); ESI-MS (positive mode): m/z = 1025 (M + Na)⁺. ESI-MS/MS (positive mode): m/z = 1025 [M + Na]⁺ → 863 [M + Na - Glc]⁺, 863 → 701 [M + Na - 2 Glc]⁺, 701 → 541 [M + Na - 2 Glc - Thev]⁺, 541 → 374 [M + Na - 2 Glc - Thev - Cym - Na]⁺; ¹H-NMR (500 MHz, C₅D₅N): δ = 0.98 (3H, s, H-18), 0.87 (3H, s, H-19), 1.62 (3H, d, J = 8.3 Hz, H-6′), 1.67 (3H, d, J = 6.2 Hz, H - 6″), 3.42 (3H, s, 3′-OMe), 3.61 (3H, s, 3″-OMe), 6.12 (1H, br.s, H-22), 5.29 and 5.01 (each 1H,br.d, J = 18.0, 2.0 Hz, H-21), 4.95 (1H, br.d, J = 7.6 Hz, H-1″″), 5.07 (1H, br.d, J = 7.2 Hz, H-1′″), 5.06 (1H, br.d, J = 8.2 Hz, H-1″), 5.18 (1H, dd, J = 9.6, 2.0 Hz; H-1′); ¹³C-NMR data see Table [1].

Extensumside B (2): White amorphous powder, C₅₃H₈₄O₂₂S; m. p. 196 - 198 °C; [α]_D ²⁵: -12.0 (c 0.4, CH₃OH); IR (KBr): ν _max=3434, 2931, 1721, 1647, 1454, 1380, 1228, 1146, 1060, 1007, 589 cm^-1; UV (MeOH): λ_max (log ε) = 220 (1.13), 295 (0.23), 305 (0.21) nm. HRFABMS (negative mode): m/z = 1103.5312 [M - H]^- (calcd.: 1103.5274); ESI-MS/MS (negative mode): m/z = 1103 [M - H]^- → 1020 [M - H - 83]^-, 1020 → 876 [M - H - 83 - Cym]^-, 876 → 732 [M - H - 83 - 2 Cym]^-,732 → 573 [M - 83 - 2 Cym - Thev]^-, 1103 → 1003 [M - H₂O - 83]^-, 1003 → 903 [M - H - 2 H₂O - 83 - 81]^-, 903 → 759 [M - H - 2 H₂O - Cym - 83 - 81]^-, 759 → 615 [M - H - 2 H₂O - 2 Cym - 83 - 81]^-; ¹H-NMR (500 MHz, C₅D₅N): δ = 0.55 (3H, s, H-18), 0.81 (3H, s, H-19), 2.42 (3H, s, H-21), 2.19 (3H, s, H-d), 1.72 (3H, s, H-e), 1.23 (3H, d, J = 6.7 Hz, H-6″), 1.63 (3H, d, J = 6.6 Hz, H-6′″), 1.61 (3H, d, J = 6.6 Hz, H-6″″), 3.92 (3H, s, 3″-OMe), 3.57 (3H, s, 3′″-OMe), 3.39 (3H, s, 3″″-OMe), 4.05 (1H, m, H-3), 3.01 (1H, br.s, H-17), 5.25 (1H, br.s, H-6), 5.81 (1H, br.s, H-b), 4.81 (1H, br.d, J = 9.0 Hz; H-1′), 4.79 (1H, br.d, J = 8.5 Hz, H-1″), 5.06 (1H, dd, J = 9.6, 2.0 Hz; H-1′″), 5.30 (1H, dd, J = 9.6, 2.0 Hz; H-1″″); ¹³C-NMR data see Table [1].

Cytotoxicity assays

Eight cell lines (from ATCC) were grown in RPMI 1640 culture medium containing 5 % fetal bovine serum and 5 mM L-glutamine. The cells were harvested from exponential-phase maintenance, then cultured and dispensed on 96-well plates in 100 μL volumes. Cells in the 96-well plates were preincubated for 24 h and then treated with or without fractions of EtOH extract and the two saponins at concentrations of 0.01 - 100 μg/mL for 48 h. Taxol was used as positive control. The cytotoxicity was evaluated by the sulforhodamine B (SRB) protein assay. The GI₅₀ value (the drug concentration required to inhibit cell growth by 50 %) was used as a parameter for cytotoxicity [5], [6].

Results and Discussion

Extensumside A (1) was isolated as a white amorphous powder. It turned red when reacted with Libermann-Burchard and Kedde reagents. The pseudomolecular ion peak at m/z = 1001.4986 [M - H]^- in the HRFABMS gave the molecular formula of C₄₉H₇₈O₂₁. The IR absorption at ν_max=1737 cm^-1 and ¹³C-NMR signal at δ = 174.7 indicated one lactone carbonyl group. The ¹H-NMR signals at δ = 6.12 (br.s, H-22), 5.29 and 5.01 (each 1H, br.d, J = 18.0, 2.0 Hz, H-21), 2.75 (m, H-17), 0.98 (3H, s, H-18), 0.87 (3H, s, H-19) are characteristic for cardiac glycosides. Acidic hydrolysis [7] of 1 afforded glucose (Glc), thevetose (Thev), cymarose (Cym), and 17β-uzarigenin (1a). The sugars were identified by comparisons with authentic samples on TLC. 17β-Uzarigenin was identified by the ion peak at m/z = 373 [M - H]^- in its ESIMS and by comparison of its ¹H- and ¹³C-NMR data with those reported [8]. Four ¹H-NMR signals at δ = 1.67 (d, 3H, J = 8.3 Hz), 1.62 (d, 3H, J = 6.2 Hz), 3.61 and 3.42 (each 3H, s) confirmed the presence of thevetose and cymarose.

Besides the signals of the aglycone, the ¹H-NMR spectrum showed four anomeric protons at δ = 4.95 (1H, br.d, J = 7.6 Hz), 5.07 (1H, br.d, J = 7.2 Hz), 5.06 (1H, br.d, J = 8.2 Hz) and 5.18 (1H, dd, J = 9.2, 2.0 Hz), representing β-configurated sugar moieties. In the ESI-MS/MS, the fragmentations at m/z = 1025 [M + Na]⁺ → 863 [M + Na - Glc]⁺, 863 → 701 [M + Na - 2 Glc]⁺, 701 → 541 [M + Na - 2 Glc - Thev]⁺, 541 → 374 [M - 2 Glc - Thev - Cym]⁺ indicated the successive elimination of two glucoses, one thevetose and one cymarose moieties.

The ¹³C-NMR resonance of C-14 in 1 at δ = 84.8 resembled that in 1a at 85.0, indicating a free 14-OH group. The oligosaccharide chain could be located at C-3. The HMBC correlation of H-1′ (δ = 5.18, 1H, d, J = 8.0 Hz) in Cym′ and C-3 (δ = 73.6, d), confirmed this conclusion. Thus, from the mass spectrum, the oligosaccharide chain Glc-Glc-Thev-Cym or (Glc)₂-Thev-Cym could be deduced. The linkage of Thev and Cym could only be 1→4. H-1′ (δ = 5.18) and H-6′ (δ = 1.62, 3H, d, J = 8.3 Hz) correlated with C-5′ (δ = 69.6, d) in an HMBC experiment, suggesting that the signal for the methyl group at δ = 1.67 (3H, d, J = 6.2 Hz) could be assigned to H-6″ in Thev. The HMBC correlation of the anomeric proton at δ = 5.06 (br.d, J = 8.2 Hz) and H-6″ with C-5″ (70.6, d) suggested that this proton was H-1″ of Thev″. From the HMBC correlation of H-6″ and the proton at δ = 5.07 (1H, d, J = 7.2 Hz) with C-4″ (δ = 76.2, d), the ¹H-NMR signal at δ = 5.07 could thus be assigned to H-1′″ and a 1→ 4 linkage of Glc′″ and Thev″ could be concluded. Consequently, the ¹H-NMR signal at δ = 4.95 (1H, d, J = 7.6 Hz) could be assigned to H-1″″. The connectivity of Glc″″ and Glc′″ was 1→ 6 in view of the HMBC correlation between H-1″″ (δ = 4.95) and C-6′″ (δ = 70.6, t). Based on the evidence mentioned above, the structure of 1 was therefore established as 17β-uzarigenin 3-O-β-glucopyranosyl-(1→ 6)-β-glucopyranosyl-(1→ 4)-β-thevetopyranosyl-(1→ 4)-β-cymaropyranoside (extensumside A).

Extensumside B (2) was isolated as white amorphous powder. The Libermann-Burchard and Molish tests suggested a steroidal compound. The molecular formula C₅₃H₈₄O₂₂S was provided by the ion peak at m/z = 1103.5312 [M - H]^- in the HRFABMS. Hydrolysis of compound 2 with acid afforded glucose, thevetose and cymarose which were identified by comparison with authentic samples on TLC. In the ESI-MS/MS, the fragmentations m/z = 1103 [M - H]^- → 1020 [M - H - 83]^-, 1020 → 876 [M - H - 83 - Cym]^-, 876 → 732 [M - H - 83 - 2 Cym]^-, 732 → 572 [M - H - 83 - 2 Cym - Thev]^- revealed the successive elimination of two cymaroses and one thevetose.

In the ¹H-NMR spectrum, five methyls resonated at δ = 0.55 (s, H-18), 0.81 (s, H-19), 1.72 (s, H-d), 2.19 (s, H-e), and 2.42 (s, H-21). Two carbonyls could be recognized from ¹³C-NMR signals at 166.0 (s, C-a) and 208.6 (s, C-20), and IR absorptions at ν _max=1632 and 1721 cm^-1.

The HMBC correlations of protons at δ = 2.19 (s, H-d), and δ = 1.72 (s, H-e) with C atoms at δ = 116.1 (d, C-b), δ = 158.3 (s, C-c), and of protons at δ = 5.81 (s, H-b) with carbons at δ = 27.3 (q, C-e), δ = 20.4 (q, C-d), δ = 166.0 (s, C-a), δ = 158.3 (s, C-c) demonstrated the existence of the 3-methylbut-2-enoyl unit, which was confirmed by an ion peak at m/z = 1020 [M - H - 83]^- in the ESI-MS. This moiety could be located at C-12 according to the HMBC correlations of H-12 (δ = 3.85, m) and H-17 (δ = 3.01) with C-18 (δ = 14.7), and of H-12 with C-a (δ = 166.0, s).

In the ¹H-NMR spectrum, besides signals for methyl groups at sugar moieties, another four anomeric protons resonated at δ = 4.79 (1H, br.d, J = 8.5 Hz ), 4.81 (1H, br.d, J = 9.0 Hz ), 5.06 (1H, dd, J = 9.6, 2.0 Hz ), and 5.30 (1H, dd, J = 9.6, 2.0 Hz ). The anomeric proton at δ = 4.81 was assigned to H-1′ because of the HMBC correlation between H-1′ and C-3 (δ = 78.2). In a TOCSY experiment, the anomeric proton at δ = 4.79 (1H, br.d, J = 8.5 Hz) was grouped with the signal at δ = 1.23 (3H, d, H-6″), indicating that H-1″ resonated at δ = 4.79. Thus the ¹H-NMR signals at δ = 5.06 (1H, dd, J = 9.6, 2.0 Hz) and 5.30 (1H, dd, J = 9.6, 2.0 Hz) belonged to anomeric protons in cymarose moieties. The HMBC correlation of H-6″ (δ = 1.23) and H-1′″ (δ = 5.06, dd, J = 9.6, 2.0 Hz) with C-4″ (δ = 83.5) suggested a 1→ 4 linkage of Cym′″ and Thev″. The 1→ 4 linkage of Thev″ and Glc′ could be concluded on the basis of the HMBC correlation of H-1″ (δ = 4.79) and H-6′ (δ = 4.27) with C-4′ (δ = 81.7). From the molecular formula and ion peak at m/z = 903 [M - H - 2 H₂O - 83 - 81], a sulfonyl unit was established, which could be located at C-6′ (67.7, t) considering the downfield shift of C-6′ from 61.0 to 67.7 [9].

Therefore, the structure of 2 was determined as pregnan-5-en-20-one 12-(3-methylbut-2-enoyloxy)-3-O-[β-cymaropyranosyl-(1→ 4)-β-cymaropyranosyl-(1→ 4)-β-thevetopyranosyl-(1→ 4)-(6-O-sulfo-β-glucopyranoside)] (extensumside B).

Fractions from the 90 % EtOH extract of M. extensum, as well as extensumsides A (1) and B (2) were tested for cytotoxicity against NCI-H460, COLO-205 and MDA-MB-231 cancer cells. The results (Table [2]) showed that 1 had stronger cytotoxicity whereas 2 possessed no cytotoxicity. Further study on the cytotoxicity of 1 showed that it was moderately active with GI₅₀ values between 0.296 to 0.470 μg/mL.

As reported, the minimum structural feature of cardenolides with high affinity for Na⁺,K⁺-ATPase (NKA), was a steroid ring system with a 14β-hydroxy group and 17β-unsaturated lactone ring [10], [11], [12]. Besides the structures of the aglycones, the length [11], [13], [14], and the modifications [10] of the glycosidic chains of cardenolides affected their bioactivity.

Extensumside A (1), a cardenolide with a 14β-hydroxy group, 17β-unsaturated lactone ring, and 5′-methyl sugar at C-3 displayed cytotoxicity against 8 cell lines. Extensumside B (2) possessing no 17β-unsaturated lactone, 14β-OH and 5′-methyl sugar showed no cytotoxicity. These findings agreed with the results reported.[] [*] [*]

Acknowledgements

We thank Mr. Jian Gu of Chengdu Institute of Biology, the Chinese Academy of Sciences for collecting the material and Professor Jing-Yun Cui in Xishuangbanna Tropical Botanical Garden, the Chinese Academy of Sciences for identifying the sample.

References

• 1 Delectis Florae Reipublicae Popularis Agendae Academiae Sinicae E dita. Flora Reipublicae Popularis Sinicae. Vol 63 Beijing; Science Press 1977: pp 270-2
  Search in Google Scholar
• 2 Ran X D. Zhongyao Yaohai (Dictionary of Traditional Chinese Medicine). Vol 2 Harbin; Harbin Press 1993: p 1437
  Search in Google Scholar
• 3 Sevillano L G, Melero C P, Caballero E, Tomé F, Lelièvre L G, Geering K. et al . Inotropic activity of hydroindene amidinohydrazones.  J Med Chem. 2002;  45 127-36
  CrossrefPubMedSearch in Google Scholar
• 4 Florkiewicz R Z, Anchin J, Baird A. The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na⁺,K⁺-ATPase.  J Biol Chem. 1998;  273 544-51
  CrossrefPubMedSearch in Google Scholar
• 5 Skehan P, Storing R, Scudiero D, Monk A, McMahon J, Visica D. et al . New colorimetric cytotoxity assay for anticancer-drug-screen.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 6 Rubinstein L V, Shoemaker R H, Paul K D, Tosini S, Skehan P, Scudiero D A. et al . Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines.  J Natl Cancer Inst. 1990;  82 1113-8
  CrossrefPubMedSearch in Google Scholar
• 7 Chen J J, Zhang Z X, Zhou J, Li B T. New pregnane glycosides from Sinomarsdenia incisa .  J Nat Prod. 1999;  62 829-32
  CrossrefPubMedSearch in Google Scholar
• 8 Hesham E A, Josef H, Sayed H, Elsayada E K. Cardenolide glycosides with doubly linked sugars from Gomphocarpus sinaicus .  Phytochemistry. 1993;  34 1399-402
  CrossrefPubMedSearch in Google Scholar
• 9 Maier M S, Roccatagliata A J, Kuriss A, Chludil H, Seldes A M, Pujol C A. et al . Two new cytotoxic and virucidal trisulfated triterpene glycosides from the Antarctic sea cucumber Staurocucumis liouvillei .  J Nat Prod. 2001;  64 732-6
  CrossrefPubMedSearch in Google Scholar
• 10 Abbott A J, Holoubek C G, Martin R A. Inhibition of Na⁺,K⁺-ATPase by the cardenolide 6′-O-(E-4-hydroxycinnamoyl)desglucouzarin.  Biochem Biophys Res Commun. 1998;  251 256-9
  CrossrefPubMedSearch in Google Scholar
• 11 Choiu F CK, Watson T R. Conformational factors in cardiac glycoside activity.  J Med Chem. 1985;  28 509-15
  CrossrefPubMedSearch in Google Scholar
• 12 Chen K K. Newer cardiac glycosides and aglycones.  J Med Chem. 1970;  13 1029-34
  CrossrefPubMedSearch in Google Scholar
• 13 Koike K, Bevelle C, Talapatra S K, Cordell G A, Farnsworth N R. Potential anticancer agents. V. Cardiac glycosides of Asclepias albicans (Asclepiadaceae).  Chem Pharm Bull. 1980;  28 401-5
  PubMedSearch in Google Scholar
• 14 Hyun J W, Shin J E, Lim K H, Sung M S, Park J W, Yu J H. et al . Evomonoside: the cytotoxic cardiac glycoside from Lepidium apetalum .  Planta Med. 1995;  61 294-5
  PubMedSearch in Google Scholar

Prof. Dr. Guo-Lin Zhang

Chengdu Institute of Biology

The Chinese Academy of Sciences

P. O. Box 416

Chengdu 610041

P. R. China

Phone: +86-28-8522-5401

Fax: +86-28-8522-5401

Email: zhanggl@cib.ac.cn

References

• 1 Delectis Florae Reipublicae Popularis Agendae Academiae Sinicae E dita. Flora Reipublicae Popularis Sinicae. Vol 63 Beijing; Science Press 1977: pp 270-2
  Search in Google Scholar
• 2 Ran X D. Zhongyao Yaohai (Dictionary of Traditional Chinese Medicine). Vol 2 Harbin; Harbin Press 1993: p 1437
  Search in Google Scholar
• 3 Sevillano L G, Melero C P, Caballero E, Tomé F, Lelièvre L G, Geering K. et al . Inotropic activity of hydroindene amidinohydrazones.  J Med Chem. 2002;  45 127-36
  CrossrefPubMedSearch in Google Scholar
• 4 Florkiewicz R Z, Anchin J, Baird A. The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na⁺,K⁺-ATPase.  J Biol Chem. 1998;  273 544-51
  CrossrefPubMedSearch in Google Scholar
• 5 Skehan P, Storing R, Scudiero D, Monk A, McMahon J, Visica D. et al . New colorimetric cytotoxity assay for anticancer-drug-screen.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 6 Rubinstein L V, Shoemaker R H, Paul K D, Tosini S, Skehan P, Scudiero D A. et al . Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines.  J Natl Cancer Inst. 1990;  82 1113-8
  CrossrefPubMedSearch in Google Scholar
• 7 Chen J J, Zhang Z X, Zhou J, Li B T. New pregnane glycosides from Sinomarsdenia incisa .  J Nat Prod. 1999;  62 829-32
  CrossrefPubMedSearch in Google Scholar
• 8 Hesham E A, Josef H, Sayed H, Elsayada E K. Cardenolide glycosides with doubly linked sugars from Gomphocarpus sinaicus .  Phytochemistry. 1993;  34 1399-402
  CrossrefPubMedSearch in Google Scholar
• 9 Maier M S, Roccatagliata A J, Kuriss A, Chludil H, Seldes A M, Pujol C A. et al . Two new cytotoxic and virucidal trisulfated triterpene glycosides from the Antarctic sea cucumber Staurocucumis liouvillei .  J Nat Prod. 2001;  64 732-6
  CrossrefPubMedSearch in Google Scholar
• 10 Abbott A J, Holoubek C G, Martin R A. Inhibition of Na⁺,K⁺-ATPase by the cardenolide 6′-O-(E-4-hydroxycinnamoyl)desglucouzarin.  Biochem Biophys Res Commun. 1998;  251 256-9
  CrossrefPubMedSearch in Google Scholar
• 11 Choiu F CK, Watson T R. Conformational factors in cardiac glycoside activity.  J Med Chem. 1985;  28 509-15
  CrossrefPubMedSearch in Google Scholar
• 12 Chen K K. Newer cardiac glycosides and aglycones.  J Med Chem. 1970;  13 1029-34
  CrossrefPubMedSearch in Google Scholar
• 13 Koike K, Bevelle C, Talapatra S K, Cordell G A, Farnsworth N R. Potential anticancer agents. V. Cardiac glycosides of Asclepias albicans (Asclepiadaceae).  Chem Pharm Bull. 1980;  28 401-5
  PubMedSearch in Google Scholar
• 14 Hyun J W, Shin J E, Lim K H, Sung M S, Park J W, Yu J H. et al . Evomonoside: the cytotoxic cardiac glycoside from Lepidium apetalum .  Planta Med. 1995;  61 294-5
  PubMedSearch in Google Scholar

Prof. Dr. Guo-Lin Zhang

Chengdu Institute of Biology

The Chinese Academy of Sciences

P. O. Box 416

Chengdu 610041

P. R. China

Phone: +86-28-8522-5401

Fax: +86-28-8522-5401

Email: zhanggl@cib.ac.cn