Sesquiterpenoids from the Herb of Leonurus japonicus

Two new sesquiterpenoids, (−)-(1S*,2S*,3R*)-3-ethoxycupar-5-ene-1,2-diol (1) and (−)-(1S*,4S*,9S*)-1,9-epoxybisabola-2,10-diene-4-ol (2), along with six known compounds 3−8, were isolated from the EtOH extract of the herb of Leonurus japonicus. Their structures were elucidated by physical and spectroscopic analysis. In the in vitro assays, compounds 7 and 8 showed obvious antibacterial activity against several bacteria strains, while compound 3 significantly inhibited abnormal increase of platelet aggregation induced by ADP.


Introduction
Species of the genus Leonurus (Labiatae) are widely distributed in Eurasia, from Western Europe to China [1].A number of bioactive secondary metabolites, including alkaloids [2,3], phenylethanoid OPEN ACCESS glycosides [1], iridoid glucosides [4], cyclic peptides [5], diterpenoids [6][7][8][9] and triterpenoids [10], have been reported from several plants of this genus.Leonurus japonicus (synonyms Leonurus heterophyllus) is commonly used in Chinese herbal medicine for regulating menstrual disturbance, invigorating blood circulation, diuretics, and dispel edema [11,12].In our previous study, chemical composition and antibacterial activity of essential oils from different parts of L. japonicus have been investigated [13].The result showed that the oil of the herb ("Yimucao" in Chinese) had antibacterial activity against various Gram-positive bacteria and mainly consisted of sesquiterpenes and diterpenes, while the oil of the fruit ("Chongweizi" in Chinese) mainly made up of bornyl acetate and aliphatic hydrocarbons was inactive in the antibacterial assay.In searching for bioactive natural products from L. japonicus, we carried out a continuing investigation of the ethanolic extract of "Yimucao".Two new sesquiterpenoids 1−2 and six known compounds were isolated from the EtOAc soluble portion of the ethanolic extract.This paper describes the isolation, structure elucidation, and bioassays of these isolates.

General
NMR spectra were recorded on a Bruker-AV-400 or SYS-600 spectrometers.HRESIMS were measured with Waters Synapt G 2 HDMS.IR were recorded on a Vector 22 FT-IR spectrometer.UV spectra were obtained on a Shimadzu UV-260 spectrophotometer.Optical rotations were measured with a Perkin-Elmer 341 plus.Platelet aggregation was recorded on Labor APACT-2 aggregation meter.Column chromatography was performed with silica gel (200-300 mesh, Yantai Institute of Chemical Technology, Yantai, China), MCI gel CHP 20P (75-150 μm, Mitsubishi Chemical, Co., Tokyo, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden).HPLC separation was performed on an instrument consisting of a Cometro 6000LDS pump and a Cometro 6000PVW UV/VIS detector with an Ultimate (250  10 mm) preparative column packed with C 18 (5 μm).TLC was carried out with glass precoated silica gel GF 254 plates (Qingdao Marine Chemical Inc., Qingdao, China).

Plant Material
The herb of L. japonicus ("Yimucao") was collected in May of 2012 from the field in Wenjiang District, Chengdu City, Sichuan Province, China.Plant identity was verified by Prof. Min Li (Chengdu University of TCM, Sichuan, China).A voucher specimen (SYMC-0522) was deposited at the School of Pharmacy, Chengdu University of TCM, China.

Antibacterial Activity Experiments
All bacteria were obtained from clinical samples and stored in the Department of Pharmacology of Chengdu University of TCM.The in vitro antibacterial activity was determined by the standard agar dilution method, according to NCCLS (National Committee for Clinical Laboratory Standard) [22].5 μL of cultures of test strains at the concentration of 1 × 10 6 CFU/mL were inoculated on Mueller Hinton agar containing different concentrations of the test compounds.The MIC values were determined after incubation at 35 °C for 24 h.

Platelet Aggregation Assay
SD rats were lightly anesthetized with ether.Blood was immediately taken from the femoral artery and anticoagulated with 3.8% trisodium citrate (9:1, v/v).Platelet rich plasma (PRP) was obtained by centrifugation of the whole blood at 800 g for 10 min.The precipitate of PRP was further centrifuged at 3000 g for 10 min to obtain platelet poor plasma (PPP).PRP was adjusted with PPP to about 2 × l0 8 ~ 4 × 10 8 platelets/L.Then, the platelet aggregation induced by ADP (final concentration: 0.05 mg/mL) was recorded on a dual sample aggregation meter according to Born's method [23].The antiplatelet efficacy was evaluated by comparing maximum aggregation response of the tested compound groups with that of control group.

Compound 1
showed IR absorptions for hydroxyl (3,418 cm −1 ) and olefinic (3,039 and 1,463 cm −1 ) functionalities.The molecular formula C 17 H 30 O 3 of 1, with three hydrogen deficiencies, was indicated by HR-ESI-MS and NMR data.