A new hetero dimeric terpenoid derivative, japonicaside C, from the flower buds of Lonicera japonica

Abstract A rare hetero dimeric terpenoid derivative, named japonicaside C, together with five known secoiridoid gloucosides were isolated from the flower buds of Lonicera japonica. The structures of these compounds were established on the basis of spectroscopic analyses. Japonicaside C is the first representative of a novel type of hetero dimeric terpenoid, biogenetically originated from a guaiane-type sesquiterpenoid and a secoiridoid glucoside. Anti-virus activity of the isolated compounds was evaluated in vitro.


Introduction
The flower buds of Lonicera japonica Thunb are commonly used in traditional Chinese medicine for the treatment of upper respiratory tract infections, fever, sores, diabetes mellitus and rheumatoid arthritis (Lee et al. 1998). A number of chemical constituents with diverse structures, including iridoids (Kawai et al. 1988;Mehrotra et al. 1988;Koichi et al. 2002), alkaloids (Zheng et al. 2012;Khan et al. 2016), flavonoids (Son et al. 1992), polyphenols (Chen et al. 2010) and triterpenoid saponins (Son et al. 1994) have been reported from various parts of this plant. In our research effort aimed at the anti-virus ingredients from the flower buds of L. japonica, a rare hetero dimeric terpenoid derivative, named japonicaside C, together with five known secoiridoid glucosides, L-phenylalaninosecologanin B (2) (Zheng et al. 2012), L-phenylalaninosecologanin C (3) (Zheng et al. 2012), secoxyloganin (4) , vogeloside (5) (Wang et al. 2011) and 7-epi-vogeloside (6) (Wang et al. 2011) were isolated from 70% ethanol extract of the flower buds of L. japonica ( Figure 1). Their structures were established on the basis of spectroscopic analyses. Anti-virus (respiratory syncytial virus (RSV)) activity of the isolated compounds was evaluated in vitro.

Results and discussion
Compound 1 was obtained as a yellow amorphous powder, and its molecular formula was determined as C 31 H 44 O 13 based on HR-eSI-Q-TOF-MS (m/z 625.2866 [M + H] + , calcd for 625.2860), with 10° of unsaturation. The 13 C NMR (Table S1) and DePT-135 spectra of 1 displayed 31 carbon signals, including 4 methyl groups (δ C 23.9, 21.0, 20.6 and 8.8), 6 methylenes (δ 119.2, 62.8, 38.5, 36.2, 33.4 and 31.4), 7 quaternary carbons [2 carbonyls (δ C 211.5 and 171.8), 2 oxygenated (δ 80.7 and 74.0), a tetrasubstituted double bond (δ 141.9 and 174.8)], as well as 14 methines. The NMR data of 1 showed two sets of signals, one set (part A) similar to Secologanic acid (Bi et al. 2008) and the other (part B) belonging to a sesquiterpene unit. Comparing the NMR data of part A of 1 with those of Secologanic acid (Bi et al. 2008), the only significant difference in the 13 C NMR spectra was the appearance of an oxygenated methine [5.07 (IH, d, J = 5.4, 3.6 Hz), δ C 96.2] instead of a carboxyl signal at δ C 175.9 in Secologanic acid (Bi et al. 2008). Thus, part A of 1 was deduced as shown in Figure S1.
The anti-virus activities of compounds 1-6 were evaluated in preventing the cytopathic effects (CPes) of RSV in Hep-2 cell, and cytotoxicity was measured in parallel with the determination of antiviral activity using ribavirin as a positive control. Compounds 1-6 exerted minimal cytotoxicity against Hep-2 cells (CC 50 > 500 μM). unfortunately, all compounds did not show antiviral effects on RSV in the higher concentrations of 100 μM (Figure 2).

Plant material
The dried flower buds of L. japonica were purchased from donghai county, Jiangsu, China and identified by Professor Zhou Wu (Jiangsu Kanion Pharmaceutical Co Ltd). A voucher specimen 2014AA208 was deposited in Jiangsu Kanion Pharmaceutical Co Ltd, Lianyungang, China.

Anti-virus activity assay
The anti-virus activities of 1-6, ribavirin was evaluated in vitro at doses of 100 μM as a positive control. eC 50 (50% effective concentration) value of RSV in the presence or absence of test compounds were calculated by microscopic observations of the CPe induced by the virus on Hep-2 cells. However, the activity values, eC 50 and SI (CC 50 /eC 50 ) indicated that all test compounds did not show antiviral effects on RSV in the higher concentrations of 100 μM, and the highest concentration of toxicity test was 500 μM.

Conclusions
In this study, a rare hetero dimeric terpenoid derivative, named japonicaside C, together with five known secoiridoid glucosides (2-6) were isolated from 70% ethanol extract of the flower buds of L. japonica. Their structures were established on the basis of spectroscopic analyses and comparison with literature values. Anti-virus (RSV) activity of the isolated compounds was evaluated in vitro. However, the activity values, eC 50 and SI (CC 50 /eC 50 ) indicated that all test compounds did not show antiviral effects on RSV in the higher concentrations of 100 μM, and the highest concentration of toxicity test was 500 μM.