Two Diterpenoids and a Cyclopenta[c]pyridine Derivative from Roots of Salvia digitaloids

Two new glucosides, salviadigitoside A (1) and salviatalin A-19-O-β-glucoside (2), belonging to the salviatalin type diterpenoids, and a new cyclopenta[c]pyridine, salviadiginine A (3), were isolated from the roots of Salvia digitaloids. Structures of these compounds were determined on the basis of spectroscopic analysis. In addition, compounds 1–3 were evaluated for their anti-inflammatory activity, but the results showed a weak anti-inflammatory activity.

Salvia digitaloides is a herbaceous perennial shrub native to the Chinese provinces of Guizhou, Sichuan, and Yunnan, which 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, to make them physically strong. As far as we know, fifteen diterpenoids have been reported from the roots of this plant [11,12]. In our previous study, two diterpenes with novel skeletons were isolated from the chloroform fraction of the roots of S. digitaloides, and salviatalin A showed a potent inhibitory effect on superoxide anion production in N-formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (FMLP/CB)-activated human neutrophils, as well as other anti-inflammatory effects [11]. In order to explore the anti-inflammatory effects and the diterpenoids constituents of the roots of S. digitaloides with novel structures, we continued to study the constituents of the n-butanol fraction of this plant. In this paper, we report the isolation and structural determination of two new diterpenoid glucosides salviadigitoside A (1) and salviatalinA-19-O-β-glucoside (2) together with a new cyclopenta[c]pyridine, salviadiginine A (3) (Figure 1) from the roots of S. digitaloids. In addition, the inhibitory activites on superoxide generation and elastase release by neutrophils of compounds 1-3 were also examined.

Purification and Characterization
The methanol-soluble extract of the dried roots of S. digitaloides was suspended in water and then extracted with chloroform and n-BuOH successively. The n-BuOH fraction was subjected to purification by a combination of conventional chromatographic techniques and resulted in the isolation of three new compounds (1-3).

Structural Elucidation of Compounds 1-3
Salviadigitoside A (1) was obtained as a colorless syrup and had a postitive rotation ([α] 25 D + 56.3, c 0.19, MeOH). The high resolution electrospray ionization mass spectroscopy (HRESI-MS) of 1 showed an ion peak at m/z 531.2209, which was consistent with the molecular formula of C 26 H 36 O 10 Na. This formula implied nine degrees of unsaturation. The UV spectrum of 1 showed absorption maxima at 220 and 283 nm, indicating the presence of α,β,γ,δ-unsaturated ketone moiety in the molecule. The IR spectrum showed strong absorption peaks for OH (3387 cm −1 ), carbonyl group (1728 cm −1 ) and furan ring (763 cm −1 ). The proton signals for an anomeric proton at δ 5.46 (d, J = 8.0 Hz), oxygenated methylene at δ 3.82 (1H, dd, J = 11.6, 1.4 Hz) and 3.69 (1H, dd, J = 11.6, 4.0 Hz), and oxygenated methines at δ 3.45-3.30 (4H, m) suggested the presence of one sugar moiety. In the 13 C NMR spectrum (Table 1) Figure 2). Thus, the methyl substituents at C-4 and C-10 have the αand β-orientation, respectively. Based on the above-mentioned observations, the structure of salviadigitoside A was assigned as 1.     SalviatalinA-19-O-β-glucoside (2) was also obtained as colorless needles with positive rotation ([α] 25 D + 107.8, c 0.47, MeOH). The HRESI-MS of 2 showed a ion peak at m/z 529.2053, consistent with the molecular formula of C 26 H 34 O 10 Na, implied ten degrees of unsaturation. The UV spectrum showed an absorption maximum at 251 and 313 nm, and the IR spectrum showed strong absorptions at 3383 and 1751 cm −1 for hydroxyl and carbonyl group in the molecule, respectively. The structure of 2 was similar to that of 1, according to the 1 H NMR and 13 C NMR spectral data ( Table 1). The differences observed in the 1 H NMR and 13 C NMR spectra of 2 compared with those of 1 were occurrence of signals for C-8 and C-9 at δ 137.3 and 159.3 instead of δ 160.4 and 81.4, respectively. In addition, the H-17 signal in 2 disappeared and the carbonyl signal at δ 117.0 in 1 shifted to δ 192.1 in 2, suggesting that the seven-membered C-ring of 2 was formed the α,β-unsaturated ketone group. Therefore, the structure of salviatalinA-19-O-β-glucoside was assigned as 2, which was supported by COSY, HSQC, HMBC (Table 1, Figure 1     2) suggested that the planar structure of salviadiginine A was established as 3. The relative stereochemistry of three hydroxyls located toward pseudo equatorial orientation was due to the presence of nuclear overhauser effect (NOE) correlation between H-5 and 7-CH 3 and the absence of NOE correlation between H-5/7-CH 3 and H-6 in the NOESY spectrum ( Figure 3). The absolute configuration of the 5,6,7-triol moiety was determined by application of the circular dichroic (CD) exciton chirality method [14]. The negative Cotton effect at 225 nm (Δε −1.19), the positive Cotton effect at 268 nm (Δε +2. 19) and the negative Cotton effect at 291 nm (Δε −0.12) were correlated to the 5S, 6R, 7R configuration. Thus, compound 3 was determined to be (5S,6R,7R)-6,7-dihydro-7-methyl-5Hcyclopenta[c]pyridine-5α,6α,7α-triol.

Anti-Inflammatory Activity
Neutrophils are the most abundant white blood cells in human-bodies. They play an important role in the immune defense against various diseases, and they also participate in the development of the inflammatory reaction. As a response to different stimuli, activated neutrophils secrete a series of cytotoxins, such as the superoxide anion radical (O 2− ), a precursor to other reactive oxygen species (ROS), bioactive lipids, granule proteases, and neutrophil elastase, a major contributor to destruction of tissue in chronic inflammatory disease. Suppression of the extensive or inappropriate activation of neutrophils by drugs has been suggested as a way to improve inflammatory diseases [15][16][17][18]. In the anti-inflammatory assay (Table 3), neutrophils were activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (FMLP/CB) to produce superoxide anion and elastase, which are both mediators of neutrophilic inflammation. In this study, the percentage of inhibition (Inh %) for compounds 1-3 at 10 μM concentration against FMLP/CB-induced superoxide anion generation by human neutrophils were 11.14 ± 3.27, 9.35 ± 4.85 and 7.88 ± 5.53, respectively. In addition, the percentage of inhibition (Inh %) for compounds 1-3 at 10 μM concentration in the FMLP/CB-induced elastase release assay were 11.83 ± 6.06, 11.12 ± 2.76 and 2.83 ± 4.91, respectively. Percentage of inhibition (Inh %) at 10 mM concentration. Results are presented as mean ± SEM (n = 3); * p < 0.05 compared with the control value (DMSO); a , this phosphatidylinositol-3-kinase inhibitor was used as a positive control for inhibition of superoxide anion generation and elastase release (n = 3).

General
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. 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, Rheinstetten, Germany) with TMS as the internal reference, and chemical shifts are expressed in δ (ppm). Silica gel (Merck, Germany, 70-230, 230-400 mesh) was used for column chromatography and thin layer chromatography (TLC) was conducted on pre-coated Kiesel gel 60 F 254 plates (Merck), and the spots were visualized by UV.

Plant Materials
The

Preparation of Human Neutrophils
Whole blood was obtained from healthy donors (20-30 years old) by venipuncture. Human neutrophils were isolated by means of dextran sedimentation, Ficoll-Paque centrifugation, and hypotonic lysis of erythrocytes [19]. The purified neutrophils were resuspended in a Ca 2+ -free Hank's balanced salt solution buffer at pH 7.4 and kept at 4 °C before use.

Measurement of Superoxide Anion Generation
The superoxide anion generation assay was carried out according to an established protocol [20], which monitors the reduction of ferricytochrome c inhibited by superoxide dismutase. In brief, the neutrophils were equilibrated with 0.5 mg/mL ferricytochrome c and 1 mM Ca 2+ at 37 °C for 2 min and then incubated with each test compound for 5 min. Cells were incubated with cytochalasin B (CB, 1 μg/mL) for 3 min, before activation by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 100 nM) for 10 min. FMLP/CB was used as a stimulant to activate neutrophils to produce superoxide anion. The changes in absorbance with reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioned spectrophotometer (Hitachi U-3010, Tokyo, Japan) with constant stirring. Calculations were based on differences in the reactions with and without superoxide dismutase (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm). LY294002 was used as a positive control.

Elastase Release Assays.
Elastase release was measured by degranulation of azurophilic granules in human neutrophils as described previously [21]. Neutrophils were equilibrated in an elastase substrate, MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), at 37 °C for 2 min and then incubated with test compounds for 5 min. Cells were activated by 100 nM FMLP and 0.5 μg/mL CB, and the changes in absorbance at 405 nm were monitored continuously to assay elastase release. The results were expressed as the percentage of elastase release in the FMLP/CB-activated, drug-free control system. LY294002 was used as a positive control.