Synergistic anti-malarial action of cryptolepine and artemisinins

Abstract

Background: Cryptolepine (CPE) is the major indoloquinoline isolated from the popular West African anti-malarial plant, Cryptolepis sanguinolenta. CPE possesses various pharmacological activities with potent anti-malarial activity against both chloroquine (CQ)-resistant and -sensitive strains. The search for safe and novel anti-malarial agents and combinations to delay resistance development to Plasmodium falciparum directed this work aimed at evaluating the anti-malarial interaction and safety of CPE in combination with some artemisinin derivatives.

Methods: The in vitro SYBR Green I, fluorescent-based, drug sensitivity assay using a fixed ratio method was carried out on the CQ-sensitive plasmodial strain 3D7 to develop isobolograms from three CPE-based combinations with some artemisinin derivatives. CPE and artesunate (ART) combinations were also evaluated using the Rane's test in ICR mice infected with Plasmodium berghei NK-65 strains in a fixed ratio combination (1:1) and fractions of their ED50s in order to determine the experimental ED50 (Zexp) of the co-administered compounds. Isobolograms were constructed to compare the Zexp to the Zadd.

Results: CPE exhibited promising synergistic interactions in vitro with ART, artemether and dihydroartemisinin. In vivo, CPE combination with ART again showed synergy as the Zexp was 1.02 ± 0.02, which was significantly less than the Zadd of 8.3 ± 0.31. The haematological, biochemical, organ/body weight ratio and histopathology indices in the rats treated with CPE at all doses (25, 50, 100 mg kg(-1) po) and in combination with ART (4 mg kg(-1)) showed no significant difference compared to the control group.

Conclusion: The combination of CPE with the artemisinin derivatives were safe in the rodent model and showed a synergistic anti-malarial activity in vivo and in vitro. This study supports the basis for the selection of CPE as a prospective lead compound as the search for new anti-malarial combinations continues.

Fig. 1

Cryptolepine hydrochloride

Fig. 2

Dose-response curves from the fixed-dose combinations of cryptolepine and artesunate. Ring-stage parasites were treated with fixed-dose combinations of cryptolepine and artesunate at 48 h after which analysis was done using the SYBR Green I fluorescent method. Each value represents the IC₅₀ calculated from at least three independent in vitro experiments

Fig. 3

Effects of combinations of cryptolepine with the artemisinin derivatives on Plasmodium falciparum growth in vitro (3D7 strain). Isobolograms show the effect of combinations of both cryptolepine with artemether (a), artesunate (b) and dihydroartemisinin (c). The interaction between cryptolepine and artesunate, artemether, or dihydroartemisinin against ring-stage parasites were determined using the SYBR Green I fluorescent-based drug sensitivity assay using the fixed ratio method. Each combination was set up in triplicate for 48 h. The FIC₅₀ concentrations were used in the plotting of the isobolograms

Fig. 4

Isobologram for the combination of cryptolepine and artesunate in the Rane’s anti-malarial test. The open circles and filled circles represent the experimental and theoretical ED₅₀s ± SEM, respectively

Fig. 5

Effect of cryptolepine and artesunate on the haematological indices (a Platelets b Lymphocytes) of Plasmodium berghei-infected ICR mice treated for 6 days. Values are expressed as Mean ± SEM (n=5), Asterisk indicates significance (P < 0.05), double Asterisk indicates significance (P < 0.01) and triple Asterisk indicates significance (P < 0.001) compared to controls (ANOVA) followed by Student’s Newman–Keuls multiple comparison test

Fig. 6

Photomicrograph (×100) showing histopathological slides of the spleen of cryptolepine (CPE) and/artesunate (ART, 6 mg kg⁻¹) treated rats. a Control b Artesunate 6 mg kg⁻¹ c CPE 100 mg kg⁻¹ d CPE 100 mg kg⁻¹ + ART 6 mg kg⁻¹, all treated for 3 days

Fig. 7

Photomicrograph (×100) showing histopathological slides of the livers of cryptolepine (CPE) and/or artesunate (ART, 6 mg kg⁻¹) treated Plasmodium berghei-infected mice. a Control, b ED₅₀ of ART (6 mg kg⁻¹), c ED₅₀ of CPE (10.7 mg kg⁻¹) d ED₅₀ (CPE + ART) e ½ ED₅₀ (CPE + ART) f ¼ ED₅₀ (CPE + ART)