New Anti-HBV C-Boivinopyranosyl Flavones from Alternanthera philoxeroides

C-boivinopyranosyl flavones have rarely been isolated from nature. In the search for anti-HBV (hepatitis b virus) constituents of Alternanthera philoxeroides, two new compounds, luteolin-6-C-β-d-boivinopyranosyl-3′-O-β-d-glucopyranoside (1) and chrysoeriol-6-C-β-d-boivinopyranosyl-4′-O-β-d-glucopyranoside (2), along with three known C-boivinopyranosyl flavones (compounds 3–5) were isolated. Their structures were determined by spectroscopic analyses including 1D and 2D NMR, HR-ESI-MS, IR spectra. Compounds 1, 2 and 3 showed significant anti-HBV activities through specifically inhibiting the secretion of HBsAg in HepG2.2.15.


Introduction
It is estimated that approximately 350 million people worldwide and 93 million in China alone are hepatitis b virus (HBV) carriers, causing approximately 500,000 deaths every year.
The current treatment strategies involving vaccines, interferons and nucleosides are unsatisfactory due to drug-resistance and adverse side effects [1][2][3].
For many years, herbs have been used as the Traditional Chinese Medicine for treatment of hepatitis B virus infection, such as Salvia miltiorrhiza, Rheum palmatum, Phyllanthi urinariae [4], etc. In recent years, many agents derived from botanical origin have been reported to possess anti-HBV activities, such as niranthin and nirtetralin from Phyllanthus, curcumin from Curcuma, alisol A from Alisma orientalis, oxymatrine from Sophora, etc. [5]. Botanical agents are attractive sources of new anti-HBV drugs.
Alternanthera philoxeroides is widely distributed in South China and mainly used in China for the treatment of measles, influenza, encephalitis b. Its major constituents are triterpenoid saponins, flavones, phytosterols, anthraquinones and organic acids [6]. Several oleanolic acid analogues from it were reported to possess anti-HBV activities. In this paper, two new 6-C-boivinopyranosyl flavones together with three known analogues were isolated from Alternanthera philoxeroides (Figure 1), and their anti-HBV activities were evaluated toward HepG2 2.2.15 cells.

Plant Materials
Alternanthera philoxeroides was bought from Qixin decoction pieces Co.Ltd, Hebei province, China and identified by Bin Li, Department of Pharmaceutical Chemistry, Beijing Institute of Radiation Medicine.

Anti-HBV Assay
The anti-HBV assay was performed according to the previous report [10]. The inhibition of the secretions of HBsAg and HBeAg was assayed by ELISA method; and the cytotoxicity was assessed by the MTT method.

Inhibition Assay of HBsAg and HBeAg Secretions HepG
The HepG2.2.15 cells were plated in 96-well cell plates at a density of 5ˆ10 4 cells¨mL´1 in 200 µL of DMEM medium, and routinely cultured at 37˝C under 5% CO 2 . Different concentrations of the studied compounds were supplemented to the medium after cells were plated. Control cultures received the carrier solvent (DMEM with 0.2% DMSO). Cells were grown in the presence of the studied compounds for 8 days with changing the medium on the 4th day. The suspension and the cells were separated and collected for HBsAg and HBeAg level tests immediately. The inhibiting rates (%) were calculated by comparing the treatment group with the tested compounds and the solvent control group with DMSO. The percent of inhibition (%) = [1´OD value of sample well/OD value of DMSO well]ˆ100.

MTT-Based Cytotoxicity Assay
HepG2.2.15 cells were cultured with test compounds for 8 days. After cultivation, cell proliferation was determined by MTT assay. Briefly, 10 µL of MTT (5 g¨mL´1) was added to each well and further incubated for 4 h. Then, the culture medium was removed from each well and DMSO was added to dissolve the purple formazan of MTT. The absorbance at 540 nm was read in the Multiskan MK3 (Thermo).

Conclusions
C-boivinopyranosyl flavones have rarely been isolated from nature; fewer than 20 compounds could be found by SCifinder. In this article, two new C-boivinopyranosyl flavones, along with three known compounds were isolated and identified. Compounds 1, 2, and 3 significantly blocked the secretion of HBsAg in a dose dependent manner. They inhibited HBsAg secretion respectively by 70.6% (compound 1), 74.1% (compound 2) and 67.3% (compound 3) at non-cytotoxic concentration of 129 µM (compounds 1 and 3), 127 µM (compound 2).