Ent-Abietanoids Isolated from Isodon serra

Four new ent-abietane diterpenoids, along with four known ones were isolated from the aerial parts of Isodon serra, a traditional Chinese folk medicine. The new diterpenoids were named as serrin K (1), xerophilusin XVII (2), and enanderianins Q and R (3 and 4), while the known ones were identified as rubescansin J (5), (3α,14β)-3,18-[(1-methylethane-1,1-diyl)dioxy]-ent-abieta-7,15(17)-diene-14,16-diol (6), xerophilusin XIV (7), and enanderianin P (8), respectively. Their structures were elucidated by extensive spectroscopic analysis and comparison with the literature. Compound 1 showed remarkable inhibitory activity towards NO production in LPS-stimulated RAW264.7 cells (IC50 = 1.8 μM) and weak cytotoxicity towards five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480).


Results and Discussion
The air-dried aerial parts of I. serra were extracted with 70% aqueous acetone solution (v/v), yielding crude extracts, which then were partitioned between EtOAc and H2O. The EtOAc-solubles were subjected to repeated column chromatography and then HPLC to afford four new ent-abietane diterpenoids named serrin K (1), xerophilusin XVII (2), and enanderianins Q and R (3 and 4).

Results and Discussion
The air-dried aerial parts of I. serra were extracted with 70% aqueous acetone solution (v/v), yielding crude extracts, which then were partitioned between EtOAc and H 2 O. The EtOAc-solubles were subjected to repeated column chromatography and then HPLC to afford four new ent-abietane diterpenoids named serrin K (1), xerophilusin XVII (2), and enanderianins Q and R (3 and 4).
HRESIMS established the molecular formula of compound 4 as C 21 H 32 O 4 with six degrees of unsaturation. Preliminary inspection of the NMR spectra of 4 suggested that it resembled 3, except for displaying one methoxy group (δ C 54.8, q). The 2D NMR spectra showed HMBC correlations from MeO (δ H 3.16) to C-7 (δ C 81.3, d), revealing a MeO substituent at C-7. ROESY correlations disclosed that the orientations of the substituents in 4 were the same as those of 3. Therefore, the structure of 4 was determined to be 3α,16-dihydroxy-7β-methoxy-ent-abieta-8 (14),15(17)-dien-18-al, and it was named enanderianin R.  HRESIMS established the molecular formula of compound 4 as C21H32O4 with six degrees of unsaturation. Preliminary inspection of the NMR spectra of 4 suggested that it resembled 3, except for displaying one methoxy group (δC 54.8, q). The 2D NMR spectra showed HMBC correlations from MeO (δH 3.16) to C-7 (δC 81.3, d), revealing a MeO substituent at C-7. ROESY correlations disclosed that the orientations of the substituents in 4 were the same as those of 3. Therefore, the structure of 4 was determined to be 3α,16-dihydroxy-7β-methoxy-ent-abieta-8 (14),15(17)-dien-18-al, and it was named enanderianin R.
Compounds 5-8 were identified by comparing their physical constant data with those reported in the literature. All of the above compounds are ent-abietane type diterpenoids, which are different from the previous diterpenoid types isolated from this species. Previous studies showed that the ent-kauranoids exhibited potent anti-tumor activities [3,24]. Therefore, with the aim of exploring diterpenoids with anti-tumor and anti-inflammatory activity which might be related to the wide use of I. serra in Chinese folk medicine, compounds with qualified sample quantity (>1 mg), 1 and 3-8, were subjected to the assay for cytotoxic activity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) and for the inhibitory activity of NO production in LPS-stimulated RAW264.7 cells. Among these, only compound 1 showed some cytotoxic potency (IC50 ranging from 9.4 to 20.4 μM), while compounds 3-8 exhibited no cytotoxicity (positive control, cis-platin, IC50 ranging from 1.9-18.3 μM). Moreover, compound 1 also revealed significant NO production inhibitory activity with an IC50 of 1.8 μM (positive control, MG132, IC50 = 0.2 μM).

General Information
Optical rotations were measured in MeOH on a JASCO P-1020 digital Polarimeter, whereas UV spectra data were obtained on a Shimadzu UV2401PC spectrophotometer (Shimadzu, Kyoto, Japan). A Tensor 27 spectrophotometer (Bruker, Karlsruhe, Germany) was used for scanning IR spectroscopy with KBr pellets. 1D-and 2D-NMR (δH 8.71, 7.55 and 7.19 for pyridine-d5) spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers (Bruker Biospin, Zurich, Switzerland). Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HRESIMS was performed on a VG Autospec-3000 spectrometer (VG Instruments, UK) at 70 eV.  Compounds 5-8 were identified by comparing their physical constant data with those reported in the literature. All of the above compounds are ent-abietane type diterpenoids, which are different from the previous diterpenoid types isolated from this species. Previous studies showed that the ent-kauranoids exhibited potent anti-tumor activities [3,24]. Therefore, with the aim of exploring diterpenoids with anti-tumor and anti-inflammatory activity which might be related to the wide use of I. serra in Chinese folk medicine, compounds with qualified sample quantity (>1 mg), 1 and 3-8, were subjected to the assay for cytotoxic activity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) and for the inhibitory activity of NO production in LPS-stimulated RAW264.7 cells. Among these, only compound 1 showed some cytotoxic potency (IC 50 ranging from 9.4 to 20.4 µM), while compounds 3-8 exhibited no cytotoxicity (positive control, cis-platin, IC 50 ranging from 1.9-18.3 µM). Moreover, compound 1 also revealed significant NO production inhibitory activity with an IC 50 of 1.8 µM (positive control, MG132, IC 50 = 0.2 µM).

Plant Material
The aerial parts of I. serra were collected in the E'mei Mountain, Sichuan Province, Leshan, China, in August 2008. The voucher specimen (KIB 2008091703) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, and was identified by Prof. Xi-Wen Li.

Cytotoxicity Assays
The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 • C in a humidified atmosphere with 5% CO 2 . Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of MTS (Sigma, St. Louis, MO, USA) [25]. Briefly, adherent cells (100 µL) were seeded into each well of a 96-well cell culture plate and were allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 × 10 5 cells/mL in 100 µL of 20% SDS-50% DMF after removal of 100 µL of medium. The optical density of the lysate was measured at 595 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 [26].

Nitric Oxide Production in RAW264.7 Macrophages
Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 10 5 cells/well) containing RPMI 1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO 2 at 37 • C. After 24 h pre-incubation, cells were treated with serial dilutions in the presence of 1 µg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 µL of Griess reagent (reagent A and reagent B, respectively, Sigma) to 100 µL of each supernatant from LPS (Sigma)-treated or LPSand compound-treated cells in triplicate. After 5 min of incubation, the absorbance was measured at 570 nm with a 2104 Envision multitable plate reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [27].

Determination of Cytotoxic Effects
The cytotoxicity of the tested compounds was evaluated using an MTS assay. Briefly, RAW264.7 cells, 2 × 10 5 cells/well, were seeded in 96-well plates. After 24 h incubation, cells were treated with or without test compounds at given concentrations for 18 h. Then, MTS was added to each well and the plates were kept for 4 h. The testing compounds were dissolved in DMSO, and the absorbance was read at 490 nm. Cytotoxicity was calculated by the cell viability of the cells without compounds as 100%.

Conclusions
Four new ent-abietane diterpenoids, together with four known ones, were isolated from Isodon serra, collected in the E mei Mountain of China, all of which were ent-abietane type discovered for the first time from this species. In vitro bioactive tests revealed that compound 1 exhibited some cytotoxicity against five human cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480), and showed significant inhibitory activity towards NO production with an IC 50 value of 1.8 µM.
Supplementary Materials: Supplementary materials are available online.