Anti-Mycobacterium tuberculosis activity of dichloromethane extract of Piper corcovadensis (Miq.) C. DC. roots and isolated compounds

Abstract

Tuberculosis (TB) is a major public health problem worldwide and the development of drug resistance, as multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB, contributed to the worsening of this scenario. Thus, there is an urgent need for the development of new anti-TB drugs. The objective of this research was to characterize the crude extract of the Piper corcovadensis roots (CEPC) by the ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis, in addition to isolate the chemical constituents by column chromatography and evaluate the antibacterial activity against Mycobacterium tuberculosis, as well as their cytotoxicities. Anti-M. tuberculosis activity (H37Rv and clinical isolates) was evaluated using resazurin microtiter assay plate (REMA), classical checkerboard assay and scanning electron microscopy (SEM). The cytotoxicity assay was realized on Vero cells. By UHPLC-MS/MS analysis, four pyrrolidine alkaloids were identified in the chemical composition of CEPC, namely: piperovatine, piperlonguminine, isopiperlonguminine and chingchengenamide A. Piperovatine and the fraction (90% piperlonguminine and 10% isopiperlonguminine) (FRPI) were isolated by column chromatography. For M. tuberculosis H37Rv, CEPC, piperovatine and FRPI showed a minimum inhibitory concentration (MIC) of 15.60; 7.80 and 7.80 μg/mL, and 50% cytotoxic concentration (CC50) of 55; 192 and 215 μg/mL in Vero cells, respectively. The MIC values for clinical isolates ranged between 0.98–3.90 μg/mL for CEPC, 0.48–1.95 μg/mL for piperovatine, and 0.48–3.90 μg/mL for FRPI. Futhermore, CEPC in combination with isoniazid and rifampicin showed synergism, while the piperovatine and FRPI presented synergism in combination with the rifampicin. By analyzing the SEM micrographs, it was possible to conclude that there were alterations in the morphology of the bacteria in all treatments and at the different times. Thus, it can be stated that CEPC, piperovatine, and FRPI showed promising in vitro results for the development of new drugs. Nevertheless, further studies are in progress to explain the mechanism of action and the in vivo activity is necessary to confirm their effectiveness.