Studies on the mitochondrial, immunological and inflammatory effects of solvent fractions of Diospyros mespiliformis Hochst in Plasmodium berghei-infected mice

Abstract

The use of medicinal plants in the treatment of malaria is gaining global attention due to their efficacy and cost effectiveness. This study evaluated the bioactivity-guided antiplasmodial efficacy and immunomodulatory effects of solvent fractions of Diospyros mespiliformis in mice infected with a susceptible strain of Plasmodium berghei (NK 65). The crude methanol extract of the stem of D. mespiliformis (DM) was partitioned between n-hexane, dichloromethane, ethyl acetate and methanol. Male Swiss mice (20 ± 2 g) infected with P. berghei were grouped and treated with vehicle (10 mL/kg, control), Artemether lumefantrine (10 mg/kg), 100, 200 and 400 mg/kg of n-hexane, dichloromethane, ethyl acetate and methanol fractions of D. mespiliformis for seven days. Blood was obtained for heme and hemozoin contents while serum was obtained for inflammatory cytokines and immunoglobulins G and M assessments. Liver mitochondria were isolated for mitochondrial permeability transition (mPT), mitochondrial F₁F₀ ATPase (mATPase) and lipid peroxidation (mLPO) assays. The GC-MS was used to identify the compounds present in the most potent fraction. The dichloromethane fraction had the highest parasite clearance and improved hematological indices relative to the drug control. The heme values increased, while the hemozoin content significantly (P < 0.05) decreased compared with the drug control. The highest dose of HF and MF opened the mPT pore while the reversal effects of DF on mPT, mATPase and mLPO were dose-dependent. The levels of IgG, IgM and TNFα in the DF group were significantly higher than the drug control, while the IL-1β and IL-6 values did not vary linearly with the dose. Lupeol and Stigmastan-3,5-diene were the most abundant phytochemicals in the DF. The outcome of this study showed that the DF has immunomodulatory effects in infected mice, reduced proliferation of the malaria parasite and thus protect liver cells.

Figure 1

Heme (a) and hemozoin (b) contents of mice treated with fractions of D. mespiliformis. ** = P < 0.01 vs normal control in (a); **** = P < 0.0001 vs negative control in (b).

Figure 2

Representative profile of effects of Diospyros mespiliformis solvent fractions on mitochondrial permeability transition pore opening. (A) Isolated mitochondria integrity is shown by insiginifant decrease in changes in absorbance for 12 min at 30 s interval (without calcium), susceptibility of these mitochondria to calcium induction of the mPT pore opening was revealed by significant difference in changes in absorbance (withcalcium) and reversal by spermine was shown as insignificant decrease in changes in absorbance (spermine). (B) Hexane fraction (HF) of DM dose dependently opened the mPT pore maximally at 400 mg/kg and higher than infected control. (C) DF reversed opening of the pore by malarial infection and similar results were obtained in the EF group (D). (E) Only 400 mg/kg dose of MF opened the pore more than malarial infection.

Figure 3

The ATPase activity of mitochondria isolated from the liver of experimental mice and mitochondrial lipid peroxidation results are presented in (A,B), respectively. Both infected control and control drug (Artemether lumefantrine) significantly (P < 0.001) enhanced ATPase activity while fractions of Diospyros mespiliformis significantly (P < 0.001) decreased ATPase activity (A). In (B), only DF 100, 200 mg/kg DF and 100 mg/kg EF were not significantly different from the normal control. The lipid peroxidation values of all groups were significantly lower than the negative control.

Figure 4

Immunomodulatory effects of Plasmodium infection and influence of treatment with dichloromethane fraction of Diospyros mespiliformis. (A) Serum TNFα significantly increased (P < 0.0001) maximally at 400 mg/kg of dichloromethane fraction of D. mespiliformis administration. (B) Serum interlukin (IL-1β) significantly (P < 0.0001) decreased in the DF-treated groups relative to the drug control while serum interlukin 6 (IL-6) of infected control and 100 mg/kg DF significantly (P < 0.0001) decreased relative to positive control (C). (D) IgG value increased maximally at 200 mg/kg of DF and (E) there is no significant difference in the IgM value in all the groups.

Figure 5

The GC–MS chromatogram of dichloromethane fraction of D. mespiliformis.