Two new abietane diterpenoid glycosides from Clinopodium chinense

Abstract Two new abietane diterpenoid glycosides, named clinopoditerpenes B (1) and C (2), were isolated from Clinopodium chinese. The structures of the new compounds were determined on the basis of extensive spectral analysis. Compound 1 exhibited cardioprotective effect against H2O2-induced apoptosis in H9c2 cells.


Introduction
The genus Clinopodium (Labiatae) contains about 20 species in the world. Many species of this genus are traditional medicinal plants with a wide distribution in the tropical and subtropical regions of south-east Asia . The aerial of Clinopodium chinense (Benth.) O. Kuntze, well known as 'duanxueliu' in China, has been used as a traditional folk medicine for the treatment of haematuria, trauma influenza and allergic dermatitis. Previous phytochemical investigations of C. chinense led to flavonoids (Aoshima et al. 2012), triterpenoid saponins (Liu et al. 1995;Wang et al. 2013) and phenylpropanoids (Murata et al. 2009), which showed anti-inflammatory and immunity, anti-hyperglycaemic, antitumour and antiradiation pharmacological effects (Tian et al. 2008). During our search for structurally interesting compounds from C. chinense, we have reported the isolation of diterpenes and prenylated naphthoquinoids Zhong et al. 2014). Further investigations of n-BuOH-soluble portion of the extracts from the aerial of C. chinense, resulted in the isolation of two new diterpenoid glycosides, clinopoditerpenes B (1) and C (2) (Figure 1). In this paper, we report the isolation and structure elucidation of compounds 1 and 2 and their cardioprotective effects against H 2 O 2 -induced apoptosis in H9c2 cells.  Protective effects of compound 1 against h 2 o 2 -induced h9c2 cell injury. h9c2 cells were pretreated with different concentrations of compounds 1 for 24 h before 150 mM h 2 o 2 was added into the medium. after incubation for 6 h at 37 °c, cell viability was determined by Mtt assay (expressed as the percentage of control). the data are expressed as mean ± sd from three independent experiments. Note: ## p < 0.01 vs. control; * p < 0.05 vs. h 2 o 2 -treated cell.
The cardioprotective effects of compounds 1 and 2 against H 2 O 2 -induced apoptosis in H9c2 cells were tested. Compared with the cell viability of 64.6 ± 3.0% in the model, 1 showed viability of 73.7 ± 3.9% at 12.5 μg mL −1 (Figure 2).

General experimental procedures
Optical rotation data were recorded using a Perkin-elmer 341 digital polarimeter in MeOH. uV data were obtained using a Shimadzu uV2550 spectrometer in MeOH. Ir data were recorded using an FTIr-8400S spectrometer. NMr spectra were recorded on Bruker AV III 600 MHz (the chemical shift values are presented as the δ values with TMS as an internal standard). Hr-eSI-MS spectra were measured on a LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Toyopearl HW-40C (75 μm, Tosoh Corporation, Tokyo, Japan), CHP 20P MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (100−200 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) and C-18 reversed-phase silica gel (50 μm, YMC CO., Ltd., Kyoto, Japan) were used for column chromatography. HPLC separation was performed on a CXTH LC-3000 HPLC system with a CXTH LC-3000 uV spectrophotometric detector and a YMC (250 × 10 mm) semi-preparative column packed with C18 (5 μm, YMC CO., Ltd., Kyoto, Japan) and precoated silica gel GF254 plates (Yantai chemical industry research institute, Yantai, China) were used for TLC. Sugar analysis was performed on an Agilent 6890 N GC equipped with a FID detector; injector temp. 250 °C; detector temp. 250 °C; N 2 as carrier gas. D-glucuronidase was purchased from Sinopharm Chemical reagent Co., Ltd. All solvents employed were analytical grade (Beijing Chemical Works, China).

Plant material
The

H9c2 cell protection assay
H9c2 cells at a density of 5 × 104 cells per well were cultured in DMeM media (Hyclone) supplemented with 10% foetal bovine serum (FBS, Hyclone), and L-glutamine (2 mM). Cultures were maintained at 37 °C in 5% CO 2 in a humidified incubator for 24 h. After 6 h of treatment with four different concentrations of drugs, followed by incubation with 150 mM H 2 O 2 for 2 h, 20 μL of 5 mg mL −1 MTT solution was added to each well (0.1 mg/well), and incubated for 4 h. The supernates were aspirated, and the formazan crystals in each well were dissolved in 150 μL of DMSO. The absorbance was measured at 570 nm on a microplate reader (BioTek, Vermont). The survival rate of H9c2 cells was evaluated and the inhibition (%) was expressed as the percentage of control (Sun et al. 2011).

Supporting information
Supporting Information as well as MS, NMr, uV, Ir spectra of compounds 1 and 2 is available online.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by [grant numbers 81173511 and 81374010] from the National Natural Sciences Foundation of China.