Our previous work revealed mutual and specific metabolites/pathways in artemisinin‐sensitive and ‐resistant Plasmodium berghei K173‐infected mice. In this study, we further investigated whether chrysosplenetin, a candidate chemical to prevent artemisinin resistance, can regulate these metabolites/pathways by integrating nontargeted metabolomics with 1H NMR and LC–Q‐TOF–MS/MS spectrum. The nuclear magnetic resonance method generated specifically altered metabolites in response to co‐treatment with chrysosplenetin, including: the products of glycolysis such as glucose, pyruvate, lactate and alanine; taurine, closely associated with liver injury; arginine and proline as essential amino acids for parasites; TMAO, a biomarker for dysbacteriosis and renal function; and tyrosine, which is used to generate levodopa and dopamine and may improve the torpor state of mice. Importantly, we noticed that chrysosplenetin might depress the activated glycolysis induced by sensitive parasites, but oppositely promoted the inhibited glycolysis to generate more lactate, which suppresses the proliferation of resistant parasites. Moreover, chrysosplentin possibly disturbs the heme biosynthetic pathway in mitochondria. The MS method yielded changed coenzyme A, phosphatidylcholine and ceramides, closely related to mitochondria β‐oxidation, cell proliferation, differentiation and apoptosis. These two means shared no overlapped metabolites and formed a more broader metabolic map to study the potential mechanisms of chrysosplenetin as a promising artemisinin resistance inhibitor.

This study investigated antimalarial efficacy and sensitization of chrysosplenetin against artemisinin‐resistant Plasmodium berghei K173 and potential molecular mechanism. Our data indicated a risk of artemisinin resistance because a higher parasitaemia% and lower inhibition% under artemisinin treatment against resistant parasites than those in the sensitive groups were observed. Two non‐antimalarial components, verapamil and chrysosplentin, being P‐gp inhibitors, possessed a strong efficacy against resistant parasites but it was not the case for Bcrp inhibitor novobiocin. Artemisinin‐chrysosplenetin combination improved artemisinin susceptibility of resistant P. berghei. Artemisinin activated intestinal P‐gp and Abcb1/Abcg2 expressions and suppressed Bcrp whereas chrysosplenetin reversed them. Resistant parasite infection led to a decreased haemozoin in organs or an increased heme in peripheral bloods compared with the sensitives; however, that in Abcb1‐deficient knockout (KO)‐resistant mice reversely got increased or decreased versus wild type (WT)‐resistant animals. Chrysosplenetin as well as rifampin (nuclear receptor agonist) increased the transcription levels of PXR/CAR while showed a versatile regulation on hepatic and enternal PXR/CAR in WT‐ or KO‐sensitive or ‐resistant parasites. Oppositely, hepatic and enteric NF‐κB p52 mRNA decreased conformably in WT but increased in KO‐resistant mice. NF‐κB pathway potentially involved in the mechanism of chrysosplenetin on inhibiting P‐gp expressions while PXR/CAR play a more complicated role in this mechanism.

Low solubility and bio-availability of artmisinin (ART) limit the clinical efficacy and unfortunately, the resistance of Plasmodium to ART has been gradually reported in recent years. In order to improve its dissolvebility, we therefore prepared ART-loaded poly(lactic co-glycolic acid) (PLGA) nanoparticles and characterized them. Later, the pharmacokinetic differences between ART original materials and artemisinin-loaded nanoparticles in mice infected with ART-sensitive and-resistant Plasmodium berghei K173 were investigated by orally administrated (40 mg/kg) by using a successfully developed and validated LC-MS/MS detection method. ART-loaded nanoparticles exhibited a smooth and spherical shape with average diameters of 193.80±7.65 nm. In vitro release results showed that ART-loaded nanoparticles displayed a stable sustained release effect. Meanwhile, the pharmacokinetic properties of ART-loaded nanoparticles were significantly improved when compared with the crude materials both in two groups. The AUC(0−t) significantly increased 2.91 and 2.85 folds as well as 4.03, 3.61 folds higher half-life period (t1/2) and 2.76, 3.27 folds higher maximum retention time (MRT), respectively. CL, meanwhile, declined 3.33 and 3.33 folds. These results suggested that ART-loaded nanoparticles enhanced the retention of ART in vivo and contributed to its long-lasting antimalarial effect.