Chemical Constituents from the Roots of Ranunculus ternatus and their Inhibitory Effects on Mycobacterium tuberculosis

Two new benzophenones, methyl (R)-3-[2-(3,4-dihydroxybenzoyl)-4,5-dihydroxyphenyl]-2-hydroxypropanoate (1) and n-butyl (R)-3-[2-(3,4-dihydroxybenzoyl)-4,5-dihydroxyphenyl]-2-hydroxypropanoate (2), were isolated from the roots of Ranunculus ternatus along with the two known compounds vanillic acid (3) and gallic acid (4). Their structures were elucidated by physical and spectroscopic analysis. In addition, compound 1 exhibited obvious activity against tuberculosis, while the activity of a 1:1 mixture of compound 1 plus 4 is better than that of 1 alone.


Introduction
Ranunculus ternatus Thunb. (Ranunculaceae) is mainly distributed in the Henan and Anhui region of China and has been used in traditional Chinese medicine for the treatment of tuberculosis, faucitis and neck scrofula. Several lactones [1,2], flavonoids [3,4], triterpenoids [5], glycosides [6] and two alkaloids [7] have been isolated from the roots of this plant. Our previous phytochemical investigations on the crude drug revealed benzophenones [8]. It was reported that the extracts of Radix Ranuncoli Ternati such as the organic acid fraction had antimycobacterial activity in vitro [9]. However, very few reports have looked into the active ingredient(s) with antituberculosis effects, apart from ternatolide [10]. Here we describe the isolation of two novel benzophenones (compounds 1, 2) and two known substituted benzene compounds (compounds 3, 4) from R. ternatus, structure elucidation on the novel compounds and their inhibitory activity against M. tuberculosis.
The two known compounds, vanillic acid (3) and gallic acid (4), were identified by comparison of their physical and spectroscopic data with literature values [13]. All the compounds were evaluated for in vitro anti-tuberculosis activity against M. tuberculosis H 37 Rv (Table 2). Compounds 1, 2, vanillic acid (3) and gallic acid (4) are responsible for the antimycobacterial activity observed in R. ternatus. Compound 1 (MIC = 41.67 ± 14.43 µg/mL) was the most active one. Furthermore, the activity of a 1:1 combination of compounds 1 and 4 (MIC = 20.83 ± 7.22 µg/mL) was better than that of 1 alone.

General Procedures
Optical rotations were measured on a WZZ-1 automatic polarimeter. IR spectra were obtained on a Bio-Rad FTS 6000 infrared spectrometer. HRESIMS spectra were performed on an Ionspec 7.0 T FTICR MS. 1D-and 2D-NMR spectra were recorded on a Bruker AVANCE-400 (400 MHz for 1 Hand 100 MHz for 13 C-) NMR spectrometer using TMS as an internal standard. Preparative HPLC was carried out on an ODS column (250 × 20 mm, YMC) with a CXTH LC-3000 UV-detector. Silica gel (200-300 mesh, Qingdao Ocean Chemical Group Co., Qingdao, China) and Sephadex LH-20 (Merck Co., Darmstadt, Germany) for column chromatography as well as silica gel GF254 (Qingdao Ocean Chemical Group Co. of China) for TLC were used.

Plant Material
The roots of Ranunculus ternatus were purchased from Zhangshu, Jiangxi Province, China and was identified by Associate Prof. Ke-Zhong Deng. A voucher specimen (voucher specimen No. RT1101) is deposited in the School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, China.

Extraction and Isolation
The roots of Ranunculus ternatus (25 kg) were powdered and successively extracted with 95% and 65% EtOH (100 L, 2 h × 3, respectively) under reflux for two hours and then filtered. After removal of the solvent under reduced pressure, the residue (4 kg) was suspended in water and partitioned with light petroleum ether and EtOAc successively. The remaining water soluble fraction (2,580 g) was passed through a macroporous resin column (Amberlite XAD 16) and eluted with H 2 O-EtOH  1,441, 1,366, 1,296, 1,222, 1,160, 1,115, 1,024, 889, 833

Anti-Tuberculosis Activity
Anti-mycobacterial activities of compounds 1-4 against M. tuberculosis strains H 37 Rv (strains were obtained from the Jiangxi Province Centers for Disease Prevention and Control, Nanchang, China) were evaluated by the Microplate Alamar Blue Assay [14]. The MIC (minimum inhibitory concentration) values were determined and compared with rifampicin as a reference drug.

Conclusions
Two new benzophenones (compounds 1, 2) together with two known organic acids (compounds 3, 4) were isolated from R. ternatus Thunb. Compounds 1-4 were assayed for their anti-tuberculosis activity and the data proved that comound 1 had significant inhibitory activity, furthermore, the activity of a mixture of compounds 1 and 4 is better than that of 1 alone. Up to now, natural benzophenones that exhibited anaphylaxis, inflammatory, alpha-glucosidase and HIV inhibition activity have been reported from species of the Clusiaceae, Thymelaeaceae, Myrtaceae and Iridaceae, etc. [15][16][17][18][19]. The substituents on the phenyl ring usually are hydroxyl, methoxy and isopentene groups. Benzophenones which possess propionate ester groups with a chiral carbon were only found in R. ternatus of the Ranunculaceae. This is the first report about benzophenones as potential anti-tuberculosis inhibitors.