Anti-plasmodial and Immunomodulatory Effects of Combined Ajuga remota and Caesalpinia volkensii Leaf Extracts during Malaria Infection in Balb/c Mice

Abstract

Malaria remains one of the most devastating infectious diseases leading to approximately 216 million infections and 445,000 deaths each year. Artemisinin combined therapy remains the first line treatment for malaria for over a decade, however there are concerns about emerging resistance especially after recrudesce was observed in South East Asia. This, therefore, stresses the need to search for alternative effective antimalarial treatment remedies. Several other plants apart from Artemisia annua have been found to have comparable anti-malarial effects either as a single preparation or a combination of different plants. This study evaluated the anti-malarial and immunomodulatory activities of Ajuga remota and Caesalpinia volkensii leaf extracts in Plasmodium berghei infected Balb/c mice as single and combined therapies. Ajuga remota and Caesalpinia volkensii leaves were extracted using ethanol and petroleum ether respectively. To assess the antimalarial activity, in vitro based inhibition assays were performed using P. falciparum 3D7. The individual extracts were at 2000 μg/ml while the combined extract had eleven combinations from 0:100 to 100:0 for A. remota + C volkensii. Chloroquine was used as a control drug at 500 µg/ml. The impact of individual and combinations of these plant extracts on the immune response was evaluated in Balb/c mice infected with P. berghei. To evaluate the impact of individual and a combination of the two plant extracts, five groups of mice (n=15 each) were inoculated with 200µl of 107 P. berghei parasites. At 2, 24, 48 and 72 hours post infection; group 1 was not treated; the second group received dihydroartemisinin (DHA); the third received A. remota extract; the fourth C. volkensii while the last group received the combined extract in the ratio of 1:1. Animals were monitored for 10 days where parasitaemia was recorded on day 2, 4, 5 and 6. At days 0, 6, 8 and day 10 post-infection three mice per group were euthanized, serum and spleens samples were obtained. Levels of immunoglobulin G (IgG) and interferon-γ (IFN-γ) were determined using enzyme-linked immunosorbent assays (ELISAs). Analysis of variance and Kruskal-Wallis non-parametric test were then done. Percentage growth inhibitions obtained were, Ajuga remota 44.9%; Caesalpinia volkensii 57.9% while the highest combined concentration gave 59.12% inhibition in vitro. In vivo antimalarial activity assessed using the 4-day suppressive assay, showed that P. berghei growth in mice was suppressed by 83.66% using a combination of A. remota and C. volkensii plant extracts, while each of the extracts suppressed growth of parasites by -23.31% and 4.34% respectively and DHA by 54.2%. IgG and IFN-γ levels in the treated groups were compared to the negative control group. IgG and IFN-γ levels (p = 0.0052 and p = 0.0331 respectively) varied during different time points showing significant difference in various groups. In conclusion, individual plant extracts though inhibited P. falciparum in vitro, they were not effective in suppressing P. berghei infection in mice. A combination of A. remota and C. volkensii plant extracts was effective at suppressing parasite growth in vivo just as the conventional drug DHA. Administration of the extracts affected the IgG and IFN-γ levels as compared to the baseline levels. A. remota herbs and C. volkensii trees should be safeguard as this study provides that they are a source of an alternative malaria treatment remedy.