Two New Cyototoxic Cardenolides from the Whole Plants of Adonis multiflora Nishikawa & Koki Ito

A phytochemical investigation of the whole plants of Adonis multiflora Nishikawa & Koki Ito. resulted in the isolation and identification of two new cardenolides—adonioside A (1) and adonioside B (6)—as well as four known cardenolides: tupichinolide (2) oleandrine (3), cryptostigmin II (4), and cymarin (5). Their structures were elucidated on the basis of NMR, MS, and IR spectroscopic analyses. Compounds 1, 2, 5, and 6 showed significant cytotoxicity against six human cancer cell lines (HCT-116, HepG2, HeLa, SK-OV-3, and SK-MEL-5, and SK-BR-3).


Introduction
Cardenolides, a chemical class within the cardiac glycosides, have a five-membered lactone group in the β position at C17 [1]. The mechanisms of these compounds are known to inhibit Na + /K + -ATPase, activate the cation pump, and increase in intracellular calcium concentration through cellular output of Na + and intake of K + [2]. Because of these biological actions, cardenolides have been used in the treatment of heart failure [3]. In addition, many researchers have suggested that cardenolides may inhibit the growth of cancer cells, and have described them as anticancer agents with fewer side effects [4,5].
Cardiac glycosides were isolated from several plant families of Ranunculaceae, Scrophulariazea, Apocynaceae, and Liliaceae, along with pregnane glycosides [6]. In Korea, the Adonis family is mainly comprised of three species, A. amurensis, A. pseudoamurensis, and A. multiflora based on RAPD analysis [7,8]. Previous phytochemical studies conducted on the roots of A. amurensis, the most well-known Adonis species, have identified several cardenolides: corchoroside A, covallatoxin, cymarin, cymarol, digitoxigenin 3-O-β-D-cymaroside, k-strophanthin, and k-strophanthin-β [9]. However, little has been reported concerning the biological and phytochemical properties of A. multiflora, except a brief report [10]. We have confirmed the presence of cardenolide spots in the TLC of ethanolic extracts from whole plants of A. multiflora based on the UV absorption pattern and the colors produced by spraying with a 10% H 2 SO 4 solution and heating. Over the course of investigating cardenolides in whole plants of A. multiflora Nishikawa & Koki Ito, two new cardenolides 1 and 6 were identified and structurally determined, along with four known ones 2-5 ( Figure 1). The cardenolides were then evaluated for cytotoxity against six human cancer cell lines (HCT-116, HepG2, HeLa, SK-OV-3, SK-BR-3, and SK-MEL-5).

Results and Discussion
The EtOH extracts were partitioned into CH2Cl2, EtOAc, BuOH, and H2O fractions. Repeated SiO2 and ODS column chromatography of the CH2Cl2 and BuOH fractions resulted in the identification of two new cardenolides, named adonioside A (1) and adonioside B (6), along with four known cardenolides 2-5. The known compounds were identified as tupichinolide (2), oleandrine (3), cryptostigmin II (4), and cymarin (5) on the basis of spectroscopic analysis and the identities were confirmed by comparing their measured spectroscopic data with those reported in the literature [11][12][13][14].
Compound 1 was isolated as a white powder and showed IR absorbance bands representing OH (3384 cm −1 ), CHO (1737 cm −1 ), and C=C (1639 cm −1 ) groups. The molecular weight was determined to be 606 from the molecular ion peak m/z 605 [M − H] − in the negative FAB-MS spectrum, and a molecular formula of C32H46O11 was determined from the high-resolved molecular ion peak (

Results and Discussion
The EtOH extracts were partitioned into CH 2 Cl 2 , EtOAc, BuOH, and H 2 O fractions. Repeated SiO 2 and ODS column chromatography of the CH 2 Cl 2 and BuOH fractions resulted in the identification of two new cardenolides, named adonioside A (1) and adonioside B (6), along with four known cardenolides 2-5. The known compounds were identified as tupichinolide (2), oleandrine (3), cryptostigmin II (4), and cymarin (5) on the basis of spectroscopic analysis and the identities were confirmed by comparing their measured spectroscopic data with those reported in the literature [11][12][13][14].
Compound 1 was isolated as a white powder and showed IR absorbance bands representing OH (3384 cm´1), CHO (1737 cm´1), and C=C (1639 cm´1) groups. The molecular weight was determined to be 606 from the molecular ion peak m/z 605 [M´H]´in the negative FAB-MS spectrum, and a molecular formula of C 32 H 46 O 11 was determined from the high-resolved molecular ion peak , and a CH 3 signal at δ(H) 1.33 (d, J = 6.8 Hz, H-6 1 ), indicated that 1 was a cardiac monoglycoside with a β-diginopyranoside.
Acid hydrolysis of 1 and purification of the hydrolysate using column chromatography resulted in a sugar compound, which was identified to be a diginopyranose by direct comparison between its    All of the isolated cardenolides from A. multiflora were evaluated for cytotoxicity against six human cancer cell lines (HCT-116, HepG2, HeLa, SK-OV-3, SK-BR-3, and SK-MEL-5). As shown in Table 2, compounds 1, 2, 5, and 6 showed significant inhibition activity against HCT-116, SK-OV-3, and SK-MEL-5 cell lines with IC50 values ranging from 0.06 ± 0.02 to 7.44 ± 1.98 µM. Compound 3 showed cytotoxic effects against the HeLa cell line with an IC50 value of 8.85 ± 0.39 µM. Compound 4 showed cytotoxicity against the SK-MEL-5 cell line with an IC50 value of 1.99 ± 0.28 µM.

Plant Materials
A. multiflora Nishikawa & Koki Ito was supplied from the BMI Corporation (Uiwang, Korea) in January 2014, and was identified by professor Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Korea. A voucher specimen (KHU2014-0117) has been reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.

Cytotoxicity Assay
The cytotoxicity of cardenolides from A. multiflora was measured by a MTT colorimetric assay. Compounds were dissolved with dimethylsulfoxide (DMSO). The cells were seeded onto 96-well microplates at a density of 1 x 10 4 cells per well in 100 µL of medium each. After incubation at 37˝C in a humidified incubator for 24 h, the cells were treated with various concentrations (1, 0.1, 0.5, 1, 5, 10, 50, 100 µM) of each compound in serum-free medium for 24 h. After incubation, 50 µL of MTT (5 mg/mL in PBS) was added to each well of the plate. The cells were incubated at 37˝C for 2 h. After removal from the medium, the cells were treated with 100 µL DMSO for 5 min and optical density measured using a microplate reader (BIO-TEK Inc., Winooski, VT, USA) at 550 nm. Cell viability was calculated as a percentage of viable cells in the compound-treated group vs. the control group by the following equation: Cell viability (%) = [OD (Compound)´OD (Blank)/OD (Control)-OD (Blank)] 100.

Statistical Analysis
All experiments were performed with triplicate samples and repeated at least three times. The data are presented as means˘SD and statistical comparisons between groups were performed using 1-way ANOVA followed by Student's t-test.

Conclusions
Two new and four known cardiac glycosides were isolated from the whole plants of Adonis multiflora Nishikawa & Koki Ito using open column chromatography and were identified based on spectroscopic data analysis, including NMR and FAB-MS. For the determination of absolute configuration, acid hydrolysis was performed. As a result, compound 1 and 6 were determined to be a 16-β-acetoxystrophanthidin 3-O-β-D-digonopyranoside, named adonioside A (1) and strophanthidin 3-O-β-D-diginopyranosyl-(1Ñ4)-β-D-glucopyronoside, named adonioside B (6). In addition, the two new compounds 1 and 6 together with the two known compounds 2 and 5 showed significant cytotoxicity against six human cancer cell lines, HCT-116, HepG2, HeLa, SK-OV-3, and SK-MEL-5, and SK-BR-3, but we couldn't establish a consistent structure-activity relationship. Consequently, these four compounds 1, 2, 5 and 6 merit futher in vivo study and on normal cell lines for bioactive selectivity. These findings suggest that A. multiflora may have potential be a useful therapeutic natural source for cancer prevention.