Effect of liquiritigenin on chloroquine accumulation in digestive vacuole leading to apoptosis-like death of chloroquine-resistant P. falciparum.

BACKGROUND: Malaria remains one of the major health concerns, especially in tropical countries. Although drugs such as artemisinin-based combinations are efficient for treating Plasmodium falciparum, the growing threat from multi-drug resistance has become a major challenge. Thus, there is a constant need to identify and validate new combinations to sustain current disease control strategies to overcome the challenge of drug resistance in the malaria parasites. To meet this demand, liquiritigenin (LTG) has been found to positively interact in combination with the existing clinically used drug chloroquine (CQ), which has become unfunctional due to acquired drug resistance. PURPOSE: To evaluate the best interaction between LTG and CQ against CQ- resistant strain of P. falciparum. Furthermore, the in vivo antimalarial efficacy and possible mechanism of action of the best combination was also assessed. METHODS: The in vitro anti-plasmodial potential of LTG against CQ- resistant strain K1 of P. falciparum was tested using Giemsa staining method. The behaviour of the combinations was evaluated using the fix ratio method and evaluated the interaction of LTG and CQ by calculating the fractional inhibitory concentration index (FICI). Oral toxicity study was carried out in a mice model. In vivo antimalarial efficacy of LTG alone and in combination with CQ was evaluated using a four-day suppression test in a mouse model. The effect of LTG on CQ accumulation was measured using HPLC and the rate of alkalinization of the digestive vacuole. Cytosolic Ca2+ level, mitochondrial membrane potential, caspase-like activity, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and Annexin V Apoptosis assay to assess anti-plasmodial potential. Proteomics analysis was evaluated by LC-MS/MS analysis. RESULTS: LTG possesses anti-plasmodial activity on its own and it showed to be an adjuvant of CQ. In in vitro studies, LTG showed synergy with CQ only in the ratio (CQ: LTG-1:4) against CQ-resistant strain (K1) of P. falciparum. Interestingly, in vivo studies, LTG in combination with CQ showed higher chemo-suppression and enhanced mean survival time at much lower concentrations compared to individual doses of LTG and CQ against CQ- resistant strain (N67) of Plasmodium yoelli nigeriensis. LTG was found to increase the CQ accumulation into digestive vacuole, reducing the rate of alkalinization, in turn increasing cytosolic Ca2+ level, loss of mitochondrial potential, caspase-3 activity, DNA damage and externalization of phosphatidylserine of the membrane (in vitro). These observations indicate the involvement of apoptosis-like death of P. falciparum that might be due to the accumulation of CQ. CONCLUSION: LTG showed synergy with CQ in the ratio LTG: CQ, 4:1) in vitro and was able to curtail the IC50 of CQ and LTG. Interestingly, in vivo in combination with CQ, LTG showed higher chemo-suppression as well as enhanced mean survival time at a much lower concentrations of both the partners as compared to an individual dose of CQ and LTG. Thus, synergistic drug combination offers the possibility to enhance CQ efficacy in chemotherapy.

Liquiritigenin, isoliquiritigenin rich extract of glycyrrhiza glabra roots attenuates inflammation in macrophages and collagen-induced arthritis in rats.

Liquiritigenin (LTG) and its bioprecursor isoliquiritigenin(ISL), the main bioactives from roots of Glycyrrhiza genus are progressively documented as a potential pharmacological agent for the management of chronic diseases. The aim of this study was to evaluate the pharmacological potential of liquiritigenin, isoliquiritigenin rich extract of Glycyrrhiza glabra roots (IVT-21) against the production of pro-inflammatory cytokines from activated macrophages as well as further validated the efficacy in collagen-induced arthritis model in rats. We also performed the safety profile of IVT-21 using standard in-vitro and in-vivo assays. Results of this study revealed that the treatment of IVT-21 and its major bioactives (LTG, ISL) was able to reduce the production of pro-inflammatory cytokines (TNF-α, IL-6) in LPS-activated primary peritoneal macrophages in a dose-dependent manner compared with vehicle-alone treated cells without any cytotoxic effect on macrophages. In-vivo efficacy profile against collagen-induced arthritis in Rats revealed that oral administration of IVT-21 significantly reduced the arthritis index, arthritis score, inflammatory mediators level in serum. IVT-21 oral treatment is also able to reduce the NFкB-p65 expression as evidence of immunohistochemistry in knee joint tissue and mRNA level of pro-inflammatory cytokines in paw tissue in a dose-dependent manner when compared with vehicle treated rats. Acute oral toxicity profile of IVT-21 demonstrated that it is safe up to 2000 mg/kg body weight in experimental mice. This result suggests the suitability of IVT-21 for further study in the management of arthritis and related complications.