A New Steroidal Saponin from the Tubers of Ophiopogon japonicus and Its Protective Effect Against Cisplatin-Induced Renal Cell Toxicity

A new furostanol saponin, ophiopogonin T, was isolated from the tubers of Ophiopogon japonicus. Its structure was established by extensive spectroscopic techniques including 1D (H and C) and 2D nuclear magnetic resonance (NMR) experiments (correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC) and nuclear Overhauser effect spectroscopy (NOESY)), high-resolution electrospray ionization mass spectrometry (ESIMS), and chemical methods. Using cell-based assays, this compound was evaluated for its cytotoxic effect on cancer cell lines and its protective effect against anticancer drug-induced nephrotoxicity. Cisplatin-induced cytotoxicity in porcine kidney (LLC-PK1) cells was significantly reduced upon treatment with ophiopogonin T, without affecting human hepatoma (HepG2) cancer cell proliferation or tube formation in human umbilical vein endothelial cells (HUVECs). These results collectively reflect the beneficial effect of ophiopogonin T on the side effects of cisplatin.


Introduction
Ophiopogon japonicus Ker-Gwal.(Liliaceae) is an evergreen, sod-forming perennial plant, widely distributed in East Asia, particularly in most areas of China.The tubers of O. japonicus have been widely used for thousands of years in traditional Chinese medicine to treat cardiovascular and chronic inflammatory diseases, 1 and have been well-known as a popular soup ingredient for nourishing 'Yin' in China. 2 A wide range of pharmacological properties such as antiviral, 3 antifungal, 3 antithrombotic, 4 anti-inflammatory, 5 and hypoglycemic activities 6 have been reported for crude extracts or isolates from the tubers of O. japonicus.][9][10][11] A survey of the literature showed that these steroidal saponins possess promising cytotoxic activities 1,7 and antimyocardial ischemia effects. 12he present paper reports the isolation and structural elucidation of a new furostanol saponin, named ophiopogonin T (1) (Figure 1) from the tubers of O. japonicus.Furthermore, compound 1 was tested for its cytotoxic effect on human hepatoma (HepG2) cells, its antimetastatic effect on human umbilical vein endothelial cells (HUVECs), and its protective effect against anticancer drug-induced nephrotoxicity in porcine kidney (LLC-PK1) cells.

Plant material
The tubers of O. japonicus were collected in Miryang, Gyeongsangbuk-do, Korea, in April 2012.A voucher specimen of the material (MPS001140-1) was identified by PhD Jeong-Hoon Lee, and deposited in the Department of Herbal Crop Research, Korea Medicinal Resources Herbarium (KMRH).

Acid hydrolysis and sugar analysis of 1
Compound 1 (0.6 mg) was dissolved in 1 mol L -1 HCl (1.0 mL) and refluxed at 100 °C for 2 h.After cooling, the hydrolysate was extracted with CHCl 3 and the aqueous layer was neutralized by passage through an Amberlite IRA-7 column and then concentrated to dryness to give a residue.The residue was dissolved in anhydrous pyridine (0.5 mL) followed by the addition of L-cysteine methyl ester hydrochloride (2 mg) (Sigma, St. Louis, MO, USA).After stirring the mixture at 60 °C for 1.5 h, hexamethyldisilazane (HMDS)-trimethylchlorosilane TMCS) (2:1, 0.4 mL) was added, and the mixture was further stirred at 60 °C for 1 h.The precipitate was removed by centrifugation, and the supernatant was concentrated under N 2 stream.The residue was partitioned between hexane and H 2 O (0.1 mL each), and the hexane layer (2 µL) was analyzed by gas chromatography (GC). 11he standard monosaccharides were subjected to the same reaction and GC analysis.Derivatives of D-xylose (Xyl), L-rhamnose (Rha), D-fucose (Fuc), and D-glucose (Glc) were detected with t R values of 12.937 min (D-xylose derivative), 14.323 min (L-rhamnose derivative), 14.678 min (D-fucose derivative), and 17.342 min (D-glucose derivative).Identification of D-xylose, L-rhamnose, D-fucose, and D-glucose for 1 was performed, giving single peaks at 12.933, 14.325, 14.680, and 17.343 min, respectively.

Cytotoxic effects on cancer cell lines, tube formation assay, and protective effects against anticancer drug-induced nephrotoxicity
The cytotoxicity of samples in HepG2 cells was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. 13In brief, cells were seeded at 1 × 10 4 cells well -1 in a 96-well plate.The following day, cells were treated with various amounts of the samples and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO 2 , 95% air.Subsequently, MTT was added to each well and the plates were incubated for an additional hour at 37 °C.The absorbance of the samples at 450 nm was measured using a microplate reader.
A tube formation assay was conducted following reported methods using HUVECs. 14In brief, cells were seeded (3 × 10 2 cells well -1 ) onto a Matrigel-coated plate, medium with or without sample was added, and the plates were then incubated at 37 °C.After 24 h, cells were fixed with 4% paraformaldehyde and stained with Mayer's hematoxylin.Cell morphology changes and tubular structure formation were observed with a light microscope.
The renoprotective effect against oxidative renal cell damage was evaluated using LLC-PK1 cells as reported previously. 15In brief, LLC-PK1 cells were seeded in 96-well culture plates at 1 × 10 4 cells per well -1 and allowed to adhere for 2 h.Thereafter, the test sample and/or 25 µmol L -1 cisplatin were added to the culture medium and incubated for 24 h.Cell viability was then measured using a microplate reader.
Regarding the identification of four sugar units in compound 1, four anomeric protons at d H 4.24 (Glc), 4.30 (Fuc), 4.41 (Xyl), and 5.32 (Rha) showed HSQC correlations with four anomeric carbons at d C 104.8, 101.0, 106.5, and 101.7, respectively.The 1 H and 13 C NMR data assignable to the sugar units and the evaluation of 2D NMR (COSY, HSQC, and HMBC) suggest that the sugar moieties of 1 consist of glucose, rhamnose, fucose, and xylose. 17,18cid hydrolysis of 1 with 1 mol L -1 HCl and GC-MS analysis yielded D-xylose, L-rhamnose, D-fucose, and D-glucose. 11he relatively large coupling constants (6.5-7.5 Hz) of each anomeric proton revealed the β configuration of Xyl, Fuc, and Glc, and the broad singlet of the anomeric proton for Rha indicated the α configuration. 17,18The sequence and interglycosidic linkages between the aglycone and the four sugar units were unambiguously defined by HMBC experiments (Figure 2).The sugar sequence of Fuc, Rha, and Xyl and its linkage to C-  19,20 The chemical shift difference (D ab = d a -d b ) between the two proton signals of H-26 could be applied to assign the 25R/25S configuration in furostanol saponins. 11,21,22The difference (D ab = 0.45) in the observed chemical shifts of H-26 demonstrated the 25R configuration (D ab < 0.48 for 25R; D ab > 0.57 for 25S). 11,21,22Thus, the structure of 1 was elucidated to be 26 The applicability of medicinal uses for compound 1 was investigated using 3 cell-based assays.In the present study, we investigated the effects of compound 1 on human hepatoma HepG2 cell proliferation, HUVEC tube formation, and cisplatin-induced nephrotoxicity in LLC-PK1 cells.the tested 3 assays, compound 1 exhibited potent protective effects against cisplatininduced kidney cell damage (Figure 3a).The LLC-PK1 cell viability decreased to 60% of the control following treatment with 25 µmol L -1 cisplatin, which was recovered significantly up to 80% of the control in a dose-dependent manner following treatment with compound 1 (Figure 3a).However, compound 1 (5 to 100 µmol L -1 ) had no effect on the proliferation of HepG2 cells or tube formation of HUVECs (Figures 3b and 3c).These results reflect the beneficial effects of compound 1 on the side effects of cisplatin without affecting HepG2 cancer cell proliferation or tube formation in HUVECs.
Cisplatin-based treatments have been considered the most effective regimens for advanced/recurrent cervical cancer, however their use is often limited because of severe nephrotoxicity. 23,24In addition, it is recommended that any compounds to be used in combination with cisplatin should have no effect on the anticancer action of cisplatin.Earlier studies have shown that pseudoginsenoside F11 ameliorates cisplatin-induced nephrotoxicity without compromising its antitumor activity. 25It has also been reported that cisplatininduced LLC-PK1 cell damage is significantly decreased by

Figure 3 .
Figure 3. Biological activities of compound 1.(a) Effect of compound 1 against cisplatin-induced nephrotoxicity in LLC-PK1 cells; (b) effect of compound 1 on HepG2 proliferation; (c) effect of compound 1 on tube formation of HUVECs.*p < 0.05 compared with the cisplatin-treated value.
1 of the aglycone was ascertained by HMBC correlations between d H 4.30 (H-1' of Fuc) and d C 85.3 (C-1), d H 5.32 (H-1" of Rha) and d C 74.0 (C-2' of Fuc), and d H 4.41 (H-1''' of Xyl) and d C 86.3 (C-3' of Fuc).In addition, the HMBC cross-peaks of d H 4.24 (H-1'''' of Glc) with d C 76.0 (C-26) allowed us to identify C-26 as the glucosyl linkage site.The stereochemistry of 1 was confirmed to be the β-orientation of OH-1 and OH-3 by analysis of the nuclear Overhauser effect spectroscopy (NOESY) spectrum showing the correlations of H-1/H-3 and H-1/H-9.Moreover, the β orientation of the hydroxy group of C-22 in the aglycone moiety was determined by the hemiketal carbon signal at d C 114.1, approximately 3 to 4 ppm downfield-shifted than that of the α configuration.