Five New Iridoids from Roots of Salvia digitaloides

Five new iridoids, salvialosides A–E (compounds 1–5), together with fifty known compounds were isolated from the roots of Salvia digitaloides. The structures of the new compounds were completely elucidated using a combination of 2D NMR techniques (COSY, NOESY, HMQC and HMBC) and HR-ESI-MS analyses. The known compounds were identified by comparison of their spectroscopic and physical data with those reported in the literature.


Introduction
Salvia is the largest genus in the economically and medicinally important family Labiatae [1], due to its many interesting biological and pharmacological activities, including antitumor [2], antiallergic [3], antioxidant [4], antimicrobial [5], and antiplatelet aggregation effects [6]. Salvia digitaloides which is an herbaceous perennial shrub native to the Chinese provinces of Guizhou, Sichuan, and Yunnan, has been used in traditional Yunnan medicine. The local Tibetans soak the roots of this plant in alcohol to manufacture a special traditional health drink, claimed to make them physically strong [7]. Although the isolation of some iridoid glycosides from Salvia digitaloides had been published in the previous literature [8,9], only diterpenes were isolated from S. digitaloides in our previous study. In order to explore the constituents of the roots of S. digitaloides, we have continued to study the constituents of this plant. In this paper, we report the isolation and structural determination of five new iridoids, salvialosides A-E (compounds 1-5, Figure 1), from the roots of S. digitaloides, together with fifty known compounds.
The HMBC correlation between H-1'" and C-4" indicated the glucosylation should be at C-4" of syringate. Hence, a 6-O-(4"-O-glucopyranosyl)syringyl-8-O-acetylshanzhiside methyl ester was deduced for 1 based on the above analysis and given the trivial name salvialoside A.  13 C-NMR spectroscopic data for compounds 1-4 in CD3OD.  3 3   Salvialosides B (2) was obtained as optically active colorless amorphous powder. The HR-ESIMS at m/z 609.1791, suggested the molecular formula C26H34O15Na. All the IR, UV, and 1 H-and 13 C-NMR spectral data (Table 1) were similar to those of 6-O-syringyl-8-O-acetylshanzhiside methyl ester (31), an iridoid 1-glucoside with a syringyl group. The esterification site was determined to be at C-6 by the downfield-shifted H-6 (δ 5.52) and the HMBC correlation ( Figure 2) of H-6 (δ 5.52) with syringyl C=O (δ 166.8). The difference between 2 and 31 was the substituent on C-8. In 2, a hydroxyl substituent, instead of an acetoxy group, on C-8 was observed by the upfield-shifted C-8 (δ from 88.3 in 31 to 79.6 in 2). Despite the presence of NOE interactions (Figure 3) between H-9 and H-10/H-1, and between H-5 and H-6, the β-orientations of the syringyl, hydroxyl, and glucosyl groups were confirmed by the the presence of weak NOE correlation between H-10 and H-6/H-1/H-7(α). Consequently, the iridoid glucoside 2 was identified as salvialosides B. It is a new compound from a natural source, although it has been obtained previously by the mild hydrolysis of 6-O-syringyl-8-O-acetylshanzhiside methyl ester (31) with NaOH in methanol [9].
Salvialosides C (3) had the molecular formula C26H34O16Na determined from HR-ESIMS at peak m/z 625.1740. In the 1 H-NMR spectrum (Table 1), compared with 2, the upfield-shifted H-6 (δ from 5.52 to 3.48) and the downfield-shifted H-7 (δ from 2.34 and 1.93 to 3.73) reflected each C-6 and C-7 connected a hydroxyl group. In the 13 C-NMR spectrum (Table 1), except 11 signals for iridoid monoterpene and six signals for glucose by comparing those with known iridoid 1-glucosides, the remaining six signals at δ 55.7, 107.1 (2 × C), 120.0, 140.6, 147.8 (2 × C), and 166.8 combined with 1 H-NMR signals at δ 3.89 (6H) and 7.33 (2H) were almost identical to the syringly signals. The downfield-shifted H-6' (δ 4.39 and 4.66) suggested that the syringic acid with C-6' hydroxyl of glucose formed an ester. The HMBC correlation of glucosyl H-6' with syringyl C=O (δ 166.8) further confirmed this connection. The stereochemistry of the aglycone of 3 was determined by coupling and NOE data (Figure 3). H-3 showed a long-range coupling (1.2 Hz) with H-5 which, in turn, showed 3 J couplings of 11.2 and 4.8 Hz with H-9 and H-5, respectively. Therefore a large coupling constant of 8.0 Hz between H-6 and H-7 indicated the two protons should be oriented in a pseudo-axial direction. In addition, the significant NOE correlations between H-9 and H-1/H-5/H-7 and between H-10 and H-6 together with the absence of NOE correlations between H-5 and H-6 as well as between H-9 and H-10 suggested that the three hydroxyls as β-6-OH, α-7-OH, and β-8-OH and they all located toward pseudo-equatorial direction. Consequently, the structure of compound 3 was assigned as salvialoside C.
Salvialosides D (4), of molecular formula C26H34O16Na according to the molecular ion peak at m/z 625.1724 in HR-ESIMS, was thus indicated to be an isomer of 3. The 1 H-NMR and 13 C-NMR signals ( Table 1) were close related to those of 3 with an iridoid monoterpene, a glucose, and a syringic acid. The syringic acid formed an ester functionality with glucose by the HMBC correlation between H-6' (δ 4.44 and 4.66) of glucose and C=O (δ 167.9) of the syringyl group. The difference between 3 and 4 was the stereochemistry of C-7 and C-8. The small coupling constant of 4.4 Hz between H-6 and H-7, together with the absence of NOE correlation between H-7 and H-5/H-9 indicated was H-7 orientated in a pseudo-equatorial direction (α-face), whereas the existence of strong NOE correlations between H-10 and H-7/H-9 and the absence of NOE correlation between H-10 and H-6 suggested a,β-H-10. The absence of NOE correlation between H-5 and H-6 confirmed the α-face of H-6. The β-6-OH, β-7-OH, and α-8-OH thus established. Therefore, compound 4 was identified as 7,8-epi-salvialoside C, and named salvialoside D.
Salvialoside E (5) was yielded as colorless powder with the molecular formula C18H28O10Na by the HR-ESIMS signal at m/z 427.1582. The IR bands at 3383, 1747, 1708 cm −1 revealed the hydroxyl and carbonyl functions. The presence of an iridoid moiety and a six-membered-ring-opened iridoid moiety was deduced according to 1D and 2D NMR spectral analysis. From the 1 H-NMR (Table 2) and COSY spectra, the iridoid moiety consisted of an oxygenated ethyl unit at δ 4.25 and 4.50 (H-3) and 1.82 and 2.05 (H-4), four mutually-coupled methines at δ 3.03 (H-9), 2.43 (H-5), 4.28 (H-6), and 3.56 (H-7) along with a methyl at δ 1.50 (H-10). Compared with iridoids 1-4, the C-1 hemiacetal was oxidized to an ester (δC 174.8) and the double bond between C-3 and C-4 was reduced to a single bond (δC 67.5 and 26.4, respectively). A δ-lactone ring existed by the HMBC correlation ( Figure 2) between H-3 and C-1. This six-membered ring was verified cis-fused to a five-membered ring by the strong NOE correlation (Figure 3  The HMBC correlations of H-9' with C-1', H-9' with C-4', and H-3' with C-5' proved their attachment. The NOE correlations between H-10' and H-6' verified the two protons positioned toward α-face. Finally, the two parts were linked together by an ester functionality as H-6 showed the HMBC correlation with C-1'. Consequently, the bis-iridoid structure of 5 was established as salvialoside E.

General Information
All the chemicals were purchased from Merck KGaA (Darmstadt, Germany) unless specifically indicated. Melting points of purified compounds were determined by a Yanagimoto MP-S3 melting point measuring apparatus without correction. UV spectra were obtained on a Hitachi UV-3210 spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were recorded on a Shimadzu FTIR spectrometer Prestige-21 (Shimadzu, Tokyo, Japan). Optical rotations were measured using a Jasco DIP-370 Polarimeter (Jasco, Tokyo, Japan). Electrospray ionization (ESI) and HRESI mass spectra were recorded on a Bruker APEX II mass spectrometer (Bruker, Rheinstetten, Germany). The NMR spectra, including 1 H-NMR, 13 C-NMR, COSY, NOESY, HMBC, and HSQC experiments, were recorded on Bruker Avance 400 and AV-500 NMR spectrometers (Bruker) with TMS as the internal reference, and chemical shifts are expressed in δ (ppm). Silica gel (Merck, 70-230, 230-400 mesh) was used for column chromatography and thin layer chromatography (TLC) was conducted on pre-coated Kiesel gel 60 F254 plates (Merck), and the spots were visualized by UV.

Plant Materials
The roots of S. digitaloides were collected from in Li Jiang, Yunnan Province, People's Republic of

Conclusions
In our investigation, fifty five compounds were isolated from the roots of Salvia digitaloides, including twelve iridoid glycosides, seven diterpenoids, seven triterpenoids, four caffeic acid sugar esters, and other compounds. Among, them the salvialosides A-E (compounds 1-5) were new compounds isolated for the first time as natural products.