Diterpenoids from the Roots of Salvia yunnanensis

Abstract Two new diterpenoids, salyunnanins I and J (1 and 2), together with ten analogues, were isolated from the roots of Salvia yunnanensis. The structures of the new isolates, possessing different neo-clerodane and seco-abietane diterpenoid skeletons respectively, were elucidated on the basis of comprehensive spectroscopic data. All of the compounds were tested for the inhibitory activities against six human tumor lines in vitro, and several ones showed moderate cytotoxic activities. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-015-0080-4) contains supplementary material, which is available to authorized users.


Introduction
The genus of Salvia is a large pool of diterpenoids with structural diversity and biological properties [1][2][3]. Many diterpenoids with interesting bioactivities, such as tanshinone IIA, salvicine, neotanshinlactone, and salvinorin A, have been reported from this genus [4][5][6]. Salvia yunnanensis is a traditional Chinese herb used as the surrogate of S. miltiorrhiza (Danshen) for the treatment of various cardiovascular diseases [7]. Many bioactive abietane diterpenoids, especially a series of abietane type diterpene alkaloids, have been reported from this plant [8][9][10]. In a continuation of our research work on the diterpenoids from Salvia species, we examined the constituents of S. yunnanensis collected in Juhuacun traditional medicine market in the Yunnan province. As a result, two new diterpenoids, salyunnanins I and J (1 and 2), together with ten known analogues (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), were isolated ( Fig. 1). It's noteworthy that the structures of the new isolates were elucidated to possess neo-clerodane and seco-abietane diterpenoid skeletons respectively. All of the compounds were tested for the inhibitory activities against six human tumor lines in vitro, and several ones showed moderate cytotoxic activities. Herein, we report the isolation, structural elucidation, and the biological evaluation of all the obtained isolates.
Salyunnanin I (1) was obtained as white powder. Its molecular formula C 22 4), and seven quaternary carbons (including two olefinic ones and three lactonic carbonyls at d C 167.9, 170.0, and 173.5). Careful analysis of these 13 C NMR data indicated that the characteristic signals for a neo-clerodane diterpenoid of two quaternary carbons (d C 49.9, C-5; 40.7, C-9), a methine (d C 39.6, C-10), an oxygenated methylene (d C 71.5, C-19), and a substituted furan ring (d C 125.5, C-13; 109.8, C-14; 144.8, C-15; 141.1, C-16) were all presented. These evidence, conjugated with some same type of diterpenoid were previously isolated in our laboratory [21], suggested that compound 1 could be a neo-clerodane diterpenoid. The 1 H and 13 C NMR data (Table 1) of 1 were very similar to those of dugesin E [21], except that the methylene carbon at d C 43.4 (C-11) in dugesin E was absent, while a down-field methine at d C 75.8 was instead present in 1. The difference implied that compound 1 was a C-11 oxygenated product of dugesin E. This assumption was supported by the HR-EIMS data, and further supported by the correlation from d H 0.80 (Me-20) to d C 75.8 (C-11) in the HMBC spectrum. Other partial planar structure of 1 was identical to those of dugesin E by detailed analysis of 1 H-1 H COSY and HMBC spectra (Fig. 2). In the NOESY spectrum, the NOE correlations of Me-20 with H 2 -19, of H-19a (d H 4.44) with H-6, of H-10 with H-8, and of H-8 with H-12 indicated that the relative configurations of C-5, C-6, C-8, C-9, C-10, and C-12 of 1 were the same as dugesin E. In addition, the strong NOE contact between Me-20 and H-11 defined the a-orientation of H-11 (Fig. 2). Hence, the structure of 1 was elucidated and named salyunnanin I.
Salvia (including 700-1050 species) is the biggest genus in the medicinal important Labiatae family and widely distributed in the world [23,24]. These plants are also a rich source of various diterpenoids, especially the clerodane and abietane diterpenoids with diverse skeletons. Structurally, the clerodane diterpeoids are typical bicyclic diterpenoids, while abietane are tricyclic diterpenoids. All of the compounds were tested for their cytotoxic effects against six human cancer cell lines (Hela, NCI-H460, PC3, KB-3-1, MCF-7, and K562) using a previously described MTT method [27]. As shown in Table 2, compounds 5, 6, and 11 exhibited moderate activities.
In conclusion, totally twelve diterpenoids, including two new ones (salyunnanins I and J), were characterized from S. yunnanensis in this study. Eleven of the isolates were abietane diterpenoids, while salyunnanin I (1) was elucidated to be a neo-clerodane diterpenoid. In the bioassay of these isolates, several compounds exhibited moderate cytotoxic activities.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were detected on a Shimadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. 1D and 2D NMR spectra were recorded on DRX-500 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts (d) were expressed in ppm with reference to the solvent signals. ESIMS and HR-EIMS analysis were carried out on Waters Xevo TQS and Waters AutoSpec Premier P776 mass spectrometers, respectively. Semi-preparative HPLC was performed on an Agile 1100 HPLC with a Zorbax SB-C 18 (9.4 9 250 mm) column. Silica gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Co., Ltd., PR China), and Amphichroic RP-18 gel (40-63 lm, Merck, Darmstadt, Germany) and MCI gel (75-150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography.

Plant Material
The roots of S. yunnanensis were collected in Kunming, Yunnan Province, PR China, in October 2010. The plant was identified by Dr. En-De Liu, Kunming Institute of Botany, Kunming, PR China. A voucher specimen was deposited at the Kunming Institute of Botany with identification number 201010S01.

Cytotoxicity Assays
The  [28]. Briefly, 100 lL of adherent cells (HeLa, NCI-H460, PC3, KB-3-1, MCF-7) was seeded into each well of a 96-well cell culture plate and allowed to adhere for 24 h before test compound addition, while suspended cells (K562) were seeded just before test compound addition, both with an initial density of 1 9 10 5 cells/mL in 100 lL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with paclitaxel (Sigma) as positive control. After the incubation, 10 lL of MTT (5 mg/mL in PBS) was added to each well, and the incubation continued for 4 h at 37°C. After removing the medium, 100 lL per well of DMSO was added to dissolve the residue and the optical density was measured at 492 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC 50 value of each compound was calculated by Reed and Muench's method [27].