title: Antiplasmodial activity of Heinsia crinita (Rubiaceae) and identification of new iridoids.
abstract: BACKGROUND
  Heinsia crinita is used in traditional medicine for the treatment of febrile illness and erectile dysfunction. Its stem bark powder is found in some peripheral markets in the Democratic Republic of the Congo (DRC) as a remedy against malaria. Investigations were conducted on crude extracts of leaves, fruits and stem barks in view to validate their use and to determine which plant part possesses the best antiplasmodial properties.
  
  
  METHODS
  Different plant parts were extracted with methanol, ethanol and dichloromethane. Based on the preliminary assays, the dichloromethane extract of the stem bark was subjected to fractionation using preparative HPLC system and column chromatography. This step led to the isolation of two new iridoids which had their structures elucidated by NMR, UV, MS and FT-IR spectroscopic techniques. Extracts and pure compounds were tested in vitro against the 3D7 strain of Plasmodium falciparum. The inhibition of the parasite growth was evaluated in vitro by colorimetric method (p-LDH assay) and their cytotoxicity evaluated in vitro against the human non-cancer fibroblast cell line (WI38) through WST1 assay. The in vivo antiplasmodial activity was assessed by the inhibition of Plasmodium berghei growth in infected mice treated with the ethanol extract of H. crinita stem bark at the concentrations of 200 and 300mg/Kg/day per os, using a protocol based on the 4-d suppressive test of Peters and compared to a non-treated negative control group of mice (growth =100%). Finally the antioxidant activity of the same extract was evaluated using ABTS, DPPH and cell-based assays.
  
  
  RESULTS
  A moderate in vitro antiplasmodial activity was observed for the dichloromethane extract of the stem bark of H. crinita (IC50=29.2±1.39µg/mL) and for the two new iridoids, lamalbide 6, 7, 8- triacetate (IC50=16.39±0.43µg/mL) as well as for its aglycone lamiridosin 6, 7, 8-triacetate (IC50=0.44.56±1.12µg/mL). The ethanolic stem bark extract (200 and 300mg/kg/day, oral route) showed a moderate in vivo antimalarial activity in Plasmodium berghei-infected mice with 27.84±2.75% and 48.54±3.76% of inhibition of the parasite growth, respectively (p<0.01).). This extract displayed high cellular antioxidant activity using dichlorofluorescein-diacetate (DCFDA) on HL-60 monocytes. These crude extracts and pure compounds tested at the higher concentration of 100µg/mL did not show any cytotoxicity against WI38 cells.
  
  
  CONCLUSIONS
  The results showed that H. crinita extracts possess antimalarial activity and contain some unusual iridoids with moderate antiplasmodial activity, therefore justifying to some extent its traditional use by the local population in DRC for this purpose. This is the first report of the isolation and antiplasmodial activity of these two new iridoids.
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium; University of Kinshasa (UNIKIN), Department of Medicinal Chemistry and Pharmacognosy, School of Pharmaceutical Sciences, P.O. Box 212, Kinshasa XI, Democratic Republic of Congo.
authors.forename: P
authors.initials: P
authors.lastname: Tshisekedi Tshibangu
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium; University of Kinshasa (UNIKIN), Department of Medicinal Chemistry and Pharmacognosy, School of Pharmaceutical Sciences, P.O. Box 212, Kinshasa XI, Democratic Republic of Congo.
authors.forename: P
authors.initials: P
authors.lastname: Mutwale Kapepula
authors.affiliation: University of Kinshasa (UNIKIN), Department of Medicinal Chemistry and Pharmacognosy, School of Pharmaceutical Sciences, P.O. Box 212, Kinshasa XI, Democratic Republic of Congo.
authors.forename: M J
authors.initials: MJ
authors.lastname: Kabongo Kapinga
authors.affiliation: University of Kinshasa (UNIKIN), Department of Medicinal Chemistry and Pharmacognosy, School of Pharmaceutical Sciences, P.O. Box 212, Kinshasa XI, Democratic Republic of Congo.
authors.forename: A
authors.initials: A
authors.lastname: Tujibikila Mukuta
authors.affiliation: University of Kinshasa (UNIKIN), Department of Medicinal Chemistry and Pharmacognosy, School of Pharmaceutical Sciences, P.O. Box 212, Kinshasa XI, Democratic Republic of Congo.
authors.forename: Dibungi T
authors.initials: DT
authors.lastname: Kalenda
authors.affiliation: Laboratory of Phytochemistry, Institute of Medical Research and Medicinal Plants Studies, (IMPM), P.O. Box 13033, Yaoundé, Cameroon.
authors.forename: Alembert T
authors.initials: AT
authors.lastname: Tchinda
authors.affiliation: Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, Sart Tilman, 4000 Liège, Belgium.
authors.forename: Ange A
authors.initials: AA
authors.lastname: Mouithys-Mickalad
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium.
authors.forename: O
authors.initials: O
authors.lastname: Jansen
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Cieckiewicz
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Tits
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Angenot
authors.affiliation: Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege (ULg), Avenue Hippocrate, 15 B36, B-4000 Liège, Belgium. Electronic address: M.frederich@ulg.ac.be.
authors.forename: M
authors.initials: M
authors.lastname: Frédérich
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000032:analysis / Q000494:pharmacology / Q000627:therapeutic use; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D005638:Fruit; D018922:HL-60 Cells; D006801:Humans; D039823:Iridoids / Q000032:analysis / Q000494:pharmacology / Q000627:therapeutic use; D008288:Malaria / Q000188:drug therapy* / Q000469:parasitology; D051379:Mice; D024301:Plant Bark; D010936:Plant Extracts / Q000032:analysis / Q000494:pharmacology / Q000627:therapeutic use; D018515:Plant Leaves; D010962:Plasmodium berghei / Q000187:drug effects / Q000254:growth & development; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development; D017382:Reactive Oxygen Species / Q000378:metabolism; D019666:Rubiaceae
chemical_list: D000962:Antimalarials; D039823:Iridoids; D010936:Plant Extracts; D017382:Reactive Oxygen Species
keywords: Heinsia crinita; Lamalbide triacetate; Lamiridosin tri-acetate; Malaria; Plasmodium falciparum