Terminalia albida treatment improves survival in experimental cerebral malaria through reactive oxygen species scavenging and anti-inflammatory properties

Abstract

Background: The development of Plasmodium resistance to the last effective anti-malarial drugs necessitates the urgent development of new anti-malarial therapeutic strategies. To this end, plants are an important source of new molecules. The objective of this study was to evaluate the anti-malarial effects of Terminalia albida, a plant used in Guinean traditional medicine, as well as its anti-inflammatory and antioxidant properties, which may be useful in treating cases of severe malaria.

Methods: In vitro antiplasmodial activity was evaluated on a chloroquine-resistant strain of Plasmodium falciparum (K-1). In vivo efficacy of the plant extract was measured in the experimental cerebral malaria model based on Plasmodium berghei (strain ANKA) infection. Mice brains were harvested on Day 7-8 post-infection, and T cells recruitment to the brain, expression levels of pro- and anti-inflammatory markers were measured by flow cytometry, RT-qPCR and ELISA. Non-malarial in vitro models of inflammation and oxidative response were used to confirm Terminalia albida effects. Constituents of Terminalia albida extract were characterized by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation patterns.

Results: In vitro antiplasmodial activity of Terminalia albida was confirmed with an IC50 of 1.5 μg/mL. In vivo, Terminalia albida treatment greatly increased survival rates in P. berghei-infected mice. Treated mice were all alive until Day 12, and the survival rate was 50% on Day 20. Terminalia albida treatment also significantly decreased parasitaemia by 100% on Day 4 and 89% on Day 7 post-infection. In vivo anti-malarial activity was related to anti-inflammatory properties, as Terminalia albida treatment decreased T lymphocyte recruitment and expression of pro-inflammatory markers in brains of treated mice. These properties were confirmed in vitro in the non-malarial model. In vitro, Terminalia albida also demonstrated a remarkable dose-dependent neutralization activity of reactive oxygen species. Twelve compounds were putatively identified in Terminalia albida stem bark. Among them, several molecules already identified may be responsible for the different biological activities observed, especially tannins and triterpenoids.

Conclusion: The traditional use of Terminalia albida in the treatment of malaria was validated through the combination of in vitro and in vivo studies.

Keywords: Experimental cerebral malaria; Inflammatory response; Malaria; Oxidative stress; Terminalia albida; UHPLC-HRMS.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Antiplasmodial activity of Terminalia albida treatment in ECM model. C57BL/6 mice were infected with P. berghei ANKA and treated 2 h later with chloroquine (5 mg/kg), Terminalia albida crude extract (100 mg/kg) or water (25 mL/kg) from Day 0 to Day 3. a Mean parasite densities during infection. b Percentages of parasite suppression according to the treatment, calculated by comparison to H2O treated mice. **P < 0.005, ***P < 0.0005 compared Terminalia albida to H2O group

Fig. 2

Effect of Terminalia albida treatment on survival, weight and cerebral symptoms in ECM model. C57BL/6 mice were infected with P. berghei ANKA and treated 2 h later with chloroquine (5 mg/kg), Terminalia albida crude extract (100 mg/kg) or water (25 mL/kg) from Day 0 to Day 4. a Percentage of survival. b Percentage of weight loss at each day compared to Day 0. c Rapid murine cerebral behavior scale (RMCBS) between D3 and D7. *P < 0.05 compared Terminalia albida to H2O groups at D7. *P < 0.05 and **P < 0.005 compared Terminalia albida to H2O groups. §P < 0.05 compared Terminalia albida to chloroquine groups

Fig. 3

Effect of Terminalia albida on cell infiltration in the brain of Plasmodium berghei-infected mice. C57BL/6 mice were infected with P. berghei and treated 2 h later with chloroquine (5 mg/kg), Terminalia albida crude extract (100 mg/kg) or water (25 mL/kg) from Day 0 to Day 4. Brains were analysed by flow cytometry at D7 post-infection. Percentages of cell populations were compared between groups for CD3+ (a), CD8+ (b) and CD4+ T lymphocytes (c). *P < 0.05, **P < 0.005, ***P < 0.0005

Fig. 4

Effect of Terminalia albida treatment on the expression of pro- and anti-inflammatory markers in brains of Plasmodium berghei-infected mice. C57BL/6 mice were infected with P. berghei and treated with chloroquine (5 mg/kg), Terminalia albida (100 mg/kg) or water. Brains were harvested at D7 (for P. berghei and VEGF) or D8 post-infection (for the other markers). Gene expression was measured by RT-qPCR and cytokine levels were measured by ELISA. Gene expression levels of (a) P. berghei, VEGF, ICAM and Granzyme B; b pro-inflammatory cytokines, c other pro-inflammatory markers, and d anti-inflammatory markers. e Cytokine levels in pg/mL. *P < 0.05, **P < 0.005, ***P < 0.0005. Data presented are mean ± standard deviation

Fig. 5

Assessment of in vitro anti-inflammatory properties of Terminalia albida. Murine macrophages were activated by LPS and IFNγ in the presence or not of 10 µg/mL of Terminalia albida. Gene expression levels of a pro-inflammatory cytokines, b other pro-inflammatory markers, c pro-inflammatory signalling pathways, and d anti-inflammatory markers. e Cytokine levels measured in supernatants, in pg/mL. *P < 0.05, **P < 0.005, ***P < 0.0005. Data are presented as mean ± SD

Fig. 6

LUCS anti-oxidant assay. a Kinetics of fluorescence emission recorded under illumination for different concentrations of extract (7.5 10−3 mg/mL–1.92 mg/mL) using the LUCS assay. b Corresponding antioxidant index calculated from RFU values as follows: AI = 1000–1000 (0∫12 RFUn sample/0∫12 RFUn control)