A New Ionone Glycoside and Three New Rhemaneolignans from the Roots of Rehmannia glutinosa

A new ionone glycoside, frehmaglutoside I (1), and three new rhemaneolignans A–C (2–4) were isolated from the 95% EtOH extract of the roots of Rehmannia glutinosa. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI-MS, 1D and 2D NMR) analyses. In addition, these compounds were evaluated for their protective effects on cardiocytes impaired by doxorubicin in H9c2 cells. Among them, compounds 1–3 exhibited protective effects against DOX-induced cardiotoxicity.


Introduction
Rehmannia glutinosa, which belongs to Scrophulariaceae family, has been used as traditional Chinese herbal medicine for thousands of years. It was recorded in the Chinese medical classic "Shennong's Herba" and was thought of in China as a "top grade" herb. Previous phytochemical studies on the dried and steamed roots of R. glutinosa have led to the isolation and identification of iridoid glycosides, ionone glycosides, phenethyl alcohol glycosides, and several other components [1][2][3][4][5]. In our previous studies, three new ursane-type triterpenes glutinosalactone A-C were isolated from the leaves, a new megastigmane, rehmamegastigmane, from the fresh roots, and two new ionone glycosides frehmaglutoside G and H from the dried roots [6][7][8]. Next, the further phytochemical study was undertaken to investigate the chemical constituents of the 95% EtOH extract of the dried roots of R. glutinosa, which led to the isolation of four new compounds: frehmaglutoside I (1) and rhemaneolignan A-C (2-4) (Figure 1). Their structures were elucidated by extensive analysis of HR-ESI-MS, 1D and 2D NMR, CD data, and chemical methods. In this paper, we describe the isolation, structure elucidation, and biological evaluation of the new compounds.

Results and Discussion
Compound 1 was obtained as pale yellow crystalline powder, with a molecular formula of C22H38O9 on the basis of a [M + Na] + ion peak at m/z 469.2407 (calcd. 469.2414) in the HRESIMS. It showed IR absorptions for hydroxyl (3361 cm −1 ), methyl (2923 cm −1 ), double bond (1698 cm −1 ) and ether linkages (1074 and 1025 cm −1 ). In the 1 H-and 13 C-NMR spectra of 1 (Table 1), the signal patterns were similar to those of frehmaglutoside H [8] from R. glutinosa, except for the presence of one methoxy (δH 3.32 (3H, s, OCH3) and δC 58.0 (OCH3)). The NMR chemical shifts at the C-11 position of 1 were shifted downfield to δH 4.15 (2H, d, J = 7.0 Hz, H-11) and δC 69.1 (C-11) compared with those of frehmaglutoside H, suggesting that the methoxy was located at the hydroxymethyl group of C-11. This finding was supported by the heteronuclear multiple-bond connectivity (HMBC) correlation from H-12 (δ 3.32) to C-11 ( Figure 2).  The relative configuration of 1 was confirmed by analysis of the NOESY spectrum, the correlations between H-5 (δ 3.73) and H-15 (δ 0.95)/H-16 (δ 1.03) revealed that they were β-oriented; on the other hand, the correlations between H-5 and H-14 (δ 1.17) indicated that these protons were α-oriented respectively ( Figure 3).
The CD spectrum of 1 displayed positive Cotton effect at 204 nm and negative Cotton effect at 232 nm, which were also similar to frehmaglutoside H. Therefore the asymmetric centers of 1 had a 1R, 2R, 5S configuration. Finally, in the acid hydrolysis of 1, D-glucose was obtained as confirmed by TLC comparison with a reference sample, and the configuration was determined by measurement of the optical rotation value. On the basis of the above analysis, 1 was identified as (7E,9E)-7-[(1R,2R,5S)trihydroxy-2,6,6-trimethylcyclohexane]-9-methyipenta-7,9-dienoic-11-methoxy-5-O-β-D-glucopyranoside, and named frehmaglutoside I ( Figure 1).  , and three methoxy groups (δH 3.81 (3H, s), 3.77 (3H, s), 3.76 (3H, s)) [9]. The 13 C-NMR and DEPT spectra ( Table 2)  . These 1 H-and 13 C-NMR data implied that compound 2 should be an oxyneolignane and were similar to those of 2,2′-dimethoxy-4-(3-hydroxypropenyl)-4′-(1,2,3-trihydroxypropyI) biphenyl ether [10], except for the fact the hydroxymethyl group (C-9) in it was replaced by an ester carbonyl (δC 169.5), and one additional methoxy unit was present in compound 2. The HMBC experiment confirmed the abovementioned suggestion and placed the additional methoxyl unit at C-9 ( Figure 2). The relative configuration of 2 was determined by analysis of the coupling constants. The H-7′ possessed a β-orientation on the basis of the observed J value of 6.0 Hz between H-7′ and H-8′, indicating that H-7′ and H-8′ were in a trans-configuration [11]. The NOESY correlations from H-8′ to H-7′ further supported the conclusion (Figure 3). Thus the relative configuration for 2 was determined. In addition, the absolute configuration of C-8′ was determined to be S, as the CD spectrum showed a positive Cotton effect at 253 nm [12,13]. Correspondingly, the absolute configuration of 7′ was elucidated as R.

Plant Material
The dried roots of R. glutinosa were collected from Jiaozuo, Henan Province in China, in November 2010. The plants were identified by Prof. Chengming Dong of Henan University of TCM. A voucher specimen (No. 20101101A) has been stored in the Department of Natural Medicinal Chemistry, School of Pharmacy, Henan University of TCM, Zhengzhou, China.

Cell Culture
The rat cardiac H9c2 myocardial cells, were spontaneously immortalized ventricular rat embryo myoblasts, were purchased from Biowit Technologies (Shenzhen, China). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a water-saturated 5.0% CO2 incubator.