RESUMEN
In search of new antiviral compounds against Zika virus we conducted a bioassay-guided fractionation of bisbenzyilisoquinoline alkaloids isolated from Cissampelos sympodialis (Menispermaceae), a medicinal plant species endemic to Brazil. Six subfractions were obtained from a tertiary alkaloidal fraction of the rhizomes (TAFrz) using preparative high-performance liquid chromatography. All the subfractions were tested against Zika virus-infected Vero cells as the cellular model to evaluate cytotoxicity and antiviral effective concentrations. The results showed that three of the six TAFrz subfractions tested were active. The most active ones were the subfraction 6 (that consisted of the alkaloids methylwarifteine and warifteine present as a mixture at a ratio of 8.8:1.2 respectively) and the subfraction 5, that was later identified as warifteine, the major tertiary alkaloid of this species. Warifteine was able to significantly reduce virus titer in Zika virus-infected Vero cells with an IC50 of 2.2 µg/ml and this effect was selective (selectivity index, SI = 68.3). Subfraction 6 had an IC50 = 3.5 µg/ml and was more cytotoxic than pure warifteine, with SI = 6.14. Fraction 5 and fraction 6 were more potent in decreasing the viral titer of Zika virus-infected Vero cells than 6-methylmercaptopurine riboside (IC50 = 24.5 µg/ml and SI = 11.9), a mercaptopurine riboside with ZIKV antiviral activity used as a positive control. Our data demonstrate that alkaloids of the bisbenzylisoquinoline type may be explored as new antiviral agents or as an useful pharmacophore for investigating ZIKV antiviral activity.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Cissampelos sympodialis Eichler (Menispermaceae) is popularly used in northeastern Brazil for the treatment of respiratory diseases such as bronchitis and asthma. Despite many pre-clinical pharmacological studies, the compounds mediating the anti-asthma activity of polar extracts of Cissampelos sympodialis leaves have not been definitively identified. AIM OF THE STUDY: Aim of the study: The aim of the study was to investigate the correlation between the bioactivity of polar extracts prepared from the leaves of C. sympodialis and the chemical composition using a 1H-NMR-based metabolomics approach. MATERIAL AND METHODS: The metabolic profile of the leaf polar extract during different phenological stages of the plant was investigated using 1H NMR spectroscopy while simultaneously screening for spasmolytic activity using guinea-pig tracheal preparations. The content of the alkaloids previously implicated in the bioactivity of Cissampelos sympodialis was determined by HPLC. RESULTS: PCA analysis of the 1H NMR data discriminated the extracts from different plant phenological stages. The contents of the major alkaloids decrease (from 2 ± 0.32 µg/mL for warifteine and 1 ± 0.14 µg/mL for methylwarifteine) to undetectable levels from 90 (CsL90 extract) and 120 (CsL120) days onwards for warifteine and methylwarifteine, respectively. All six extracts relaxed the trachea pre-contracted with carbachol, but the CsF210 extract was more potent (EC50 = 74.6 ± 7.9 µg/mL) compared to both CsL90 extracts and CsL180 in the presence of functional epithelium. PLS regression analysis of 1H-NMR spectral data demonstrated that the spasmolytic activity was better correlated with signals for flavonol derivatives. CONCLUSIONS: Our data challenge the idea that warifteine and methylwarifteine mediate the spasmolytic activity of the polar extract of C. sympodialis leaves.