Bioactive Diterpenoids from Clerodendrum kiangsiense

A new abeo-abietane diterpenoid, 12-methoxy-6,11,14,16-tetrahydroxy-17(15→16)-abeo-5,8,11,13-abietatetraen-3,7-dione (8), was isolated from the hydroalcoholic extract of the herb of Clerodendrum kiangsiense along with seven known diterpenoids (1–7). Their structures were identified on the basis of spectroscopic analyses including two-dimensional NMR and comparison with literature data. All of these compounds were evaluated for their cytotoxic activities against the growth of human cancer cells lines HL-60, SMMC-7721, A-549 and MCF-7 by the MTT assay. The results showed that cryptojaponol (4), fortunin E (6) and 8 exhibited significant cytotoxicity against four human cancer cell lines.


Introduction
Abietane diterpenoids are a class of tricyclic diterpenoids, and have been isolated from plant species from the taxonomic families Verbenaceae, Lamiaceae, Taxaceae [1][2][3][4]. Clerodendrum is a genus of about 400 species in the family Verbenaceae, which mainly grows in tropical and warm temperate zones including Africa and Southern Asia. A few species are found in South America, Northern Australia and Eastern Asia [5]. Preparations of the leaves, branches and stems of Clerodendrum have been used in folk medicine to treat different diseases such as cancer, catarrhal affections of the lungs, fever, inflammation, skin diseases and asthma [6,7].  Phytochemical investigations on Clerodendrum species revealed various diterpenoids in the plants, which showed antibacterial and cytotoxic activities [8,9]. However, the phytochemical composition of the stems and roots of C. kiangsiense have not been full characterized. As a part of our ongoing effort to discover potential anticancer compounds from Chinese medicinal plants, the stems of C. kiangsiense, collected in Jiangxi Province, were investigated systematically. This has led to the isolation and structure elucidation of a new abeo-abietane diterpenoid (8) and seven known substances (1-7) (Figure 1). Compounds 1-8 were evaluated for their cytotoxicity against four cancer cell lines.

Results and Discussion
Compound 8 was isolated as colorless crystals from the EtOAc fraction of the ethanol extract of C. kiangsiense, it was assigned the molecular formula C21H26O7 (9 degrees of unsaturation) by HRESIMS (m/z 389.1597 [M − H] − , calcd. for C21H25O7 − , 389.1599). The absorption bands in the UV spectrum (238, 285, 336, 381 nm) exhibited the presence of a benzene and a ketone. In the IR spectrum, two carbonyl signals were observed at 1660 and 1715 cm −1 in addition to the absorption peak at 3405 cm −1 for the hydroxyl moiety.
To investigate their cytotoxic activities, these compounds were evaluated using an MTT cytotoxicity assay against human myeloid leukemia (HL-60), hepatocellular carcinoma (SMMC-7721), lung cancer (A-549) and breast cancer (MCF-7) cell lines. The IC 50 values of all eight compounds against the indicated cancer cells are summarized in Table 2. Compound 6 and 8 exhibited the strongest cytotoxicity to all cells, as its range of IC 50 values was 1.8-5.0 µM. Additionally, A-549 was the most sensitive cell line to these types of compounds among all tested cancer cells because the IC 50 values of all compounds against A-549 cells were close to 10 µM. Furthermore, the cytotoxicities of all the isolated compounds were comparable to the chemotherapeutic drug cisplatin [17], which suggests that these compounds might have promising potential to be anticancer agents.

General Experimental Procedures
Melting points were measured using a XT-4 micro melting point apparatus (Beijing, China). Optical rotations were determined at 25˝C on a JASCO (Tokyo, Japan) P2000 polarimeter. UV data were measured using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were recorded on a Bruker Avance III 500 spectrometer (Bruker, Bremen, Germany), using TMS as internal standard, Chemical shifts are reported as δ values and the coupling constants (J) are in Hz. HRESIMS data were obtained on a Agilent 6210 TOF-MS mass spectrometer; HPLC (Amersham Biosciences, GE Healthcare Life Science, Santa Clara, CA, USA), Waters 1525 semi-preparative HPLC system (Waters Co. Ltd., Milford, MA, USA) coupled with a Waters 2996 photodiode array detector. A Kromasil C18 preparative HPLC column (250ˆ10 mm, 5 µm) was used; Column chromatography was carried out on silica gel (Qing Dao Hai Yang Chemical Group Co., Qingdao, China; 200-300 mesh) and Sephadex LH-20 (Amersham Biosciences). TLC analyses were carried out on silica gel 60 F 254 (Merck, Darmstadt, Germany) and RP-18 F 254s (Merck) plates. Compounds were detected by UV and 30% H 2 SO 4 spraying reagent followed by heating at 105˝C for 1-2 min.

Plant Material
The stems of C. kiangsiense were collected on the Wugong Mountain of Pingxiang City, Jiangxi Province, China, in September 2010, and identified by Chunhui Dai in Zhejiang Academy of Traditional Chinese Medicine. A voucher specimen (No. 201007) has been deposited in the Key Laboratory for Genetic Improvement and Quality Control of Medical Plants of Zhejiang Province.

Anti-Proliferative Activity
The percentage of growth inhibition was determined using a MTT colorimetric technique to measure four viable cells (HL-60 human myeloid leukemia, SMMC-7721 hepatocellular carcinoma, A-549 lung cancer and MCF-7 breast cancer) with minor modification [18,19]. A total of 5000-10,000 exponential phase cells per well were seeded onto a 96-well plate for 24 h, Each tumor cell line was exposed to a test compound at concentrations of 0.0624, 0.32, 1.6, 8, and 40 µM in DMSO in triplicate for 72 h, with cisplatin as the positive control. Briefly, 100 µL of a MTT working solution (1 mg/mL) was added into each well and incubated at 37˝C for 4 h and then the medium was removed. The converted dye formazan was solubilized with 150 µL acidic isopropanol (0.04 M HCl in absolute isopropanol) and each concentration was tested in triplicate. The absorbance was then measured at a wavelength of 570 nm using a microplate reader (Multiskan Spectrum, Thermo Electron Corporation, Vantaa, Finland). The dose resulting in 50% inhibition of cell growth (IC 50 ) was calculated by the Reed and Muench method.

Conclusions
In the present study, a new abeo-abietane diterpenoid, 12-methoxy-6,11,14,16-tetrahydroxy-17(15Ñ16)-abeo-5,8,11,13-abietatetraen-3,7-dione (8) was isolated from C. kiangsiense by chromatographic separation of a 90% EtOH extract of its stems. In addition, 7 known compounds (1-7) were also obtained. The structures of compounds 1-8 were determined by spectroscopical data interpretation. The isolated compounds were subsequently evaluated for cytotoxic activities against HL-60 human myeloid leukemia, SMMC-7721 hepatocellular carcinoma, A-549 lung cancer and MCF-7 breast cancer cells, respectively. Compounds 4, 6 and 8 exhibited the strongest cytotoxicity to all cells. Additionally, A-549 was the most sensitive cell line to these types of compounds among all tested cancer cells. Furthermore, the cytotoxicities of the isolated compounds were comparable to the chemotherapeutic drug cisplatin, which suggests that C. kiangsiense and its constituents could be useful sources of candidates for the development of anticancer medicines.