title: Apoptosis mediated leishmanicidal activity of Azadirachta indica bioactive fractions is accompanied by Th1 immunostimulatory potential and therapeutic cure in vivo.
abstract: Exploration of immunomodulatory antileishmanials of plant origin is now being strongly recommended to overcome the immune suppression evident during visceral leishmaniasis (VL) and high cost and toxicity associated with conventional chemotherapeutics. In accordance, we assessed the in vitro and in vivo antileishmanial and immunomodulatory potential of ethanolic fractions of Azadirachta indica leaves (ALE) and seeds (ASE).
  
  
  
  A. indica fractions were prepared by sequential extraction of the powdered plant parts in hexane, ethanol and water. Erythrosin B staining was employed to appraise the anti-promastigote potential of ALE and ASE. Cytostatic or cytocidal mode of action was ascertained and alterations in parasite morphology were depicted under oil immersion light microscopy. Study of apoptotic correlates was performed to deduce the mechanism of induced cell death and anti-amastigote potential was assessed in Leishmania parasitized RAW 264.7 macrophages. In vivo antileishmanial effectiveness was evaluated in L. donovani infected BALB/c mice, accompanied by investigation of immunomodulatory potential of ALE and ASE. Adverse toxicity of the bioactive fractions against RAW macrophages was studied by MTT assay. In vivo side effects on the liver and kidney functions were also determined. Plant secondary metabolites present in ALE and ASE were analysed by Gas chromatography-mass spectrometry (GC-MS).
  
  
  
  ALE and ASE (500 μg ml(-1)) exhibited leishmanicidal activity in a time- and dose-dependent manner (IC₅₀ 34 and 77.66 μg ml(-1), respectively) with alterations in promastigote morphology and induction of apoptosis. ALE and ASE exerted appreciable anti-amastigote potency (IC₅₀ 17.66 and 24.66 μg ml(-1), respectively) that was coupled with profound in vivo therapeutic efficacy (87.76% and 85.54% protection in liver and 85.55% and 83.62% in spleen, respectively). ALE exhibited minimal toxicity with selectivity index of 26.10 whereas ASE was observed to be non-toxic. The bioactive fractions revealed no hepato- and nephro-toxicity. ALE and ASE potentiated Th1-biased cell-mediated immunity along with upregulation of INF-γ, TNF-α and IL-2 and decline in IL-4 and IL-10 levels. GC-MS analysis revealed several compounds that may have contributed to the observed antileishmanial effect.
  
  
  
  Dual antileishmanial and immunostimulatory efficacy exhibited by the bioactive fractions merits their use alone or as adjunct therapy for VL.
authors.affiliation: Department of Biotechnology, Parasite Immunology Laboratory, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, 110062, India. garimachouhan68@gmail.com.
authors.forename: Garima
authors.initials: G
authors.lastname: Chouhan
authors.affiliation: Department of Biotechnology, Parasite Immunology Laboratory, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, 110062, India. mdislam2009@gmail.com.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Islamuddin
authors.affiliation: Department of Biotechnology, Parasite Immunology Laboratory, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, 110062, India. muzamil_want@yahoo.com.
authors.forename: Muzamil Y
authors.initials: MY
authors.lastname: Want
authors.affiliation: Department of Biotechnology, Centre for Transgenic Plant Development, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, 110062, India. mzabdin@rediffmail.com.
authors.forename: Malik Z
authors.initials: MZ
authors.lastname: Abdin
authors.affiliation: Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box: 344, Universities Road, Medina, 30001, Saudi Arabia. hani.ozbak@gmail.com.
authors.forename: Hani A
authors.initials: HA
authors.lastname: Ozbak
authors.affiliation: Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box: 344, Universities Road, Medina, 30001, Saudi Arabia. hasanhemeg@hotmail.com.
authors.forename: Hassan A
authors.initials: HA
authors.lastname: Hemeg
authors.affiliation: Malaria Research Group, International Centre For Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. dsahal@gmail.com.
authors.forename: Dinkar
authors.initials: D
authors.lastname: Sahal
authors.affiliation: Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box: 344, Universities Road, Medina, 30001, Saudi Arabia. afrin_farhat@yahoo.co.in.
authors.forename: Farhat
authors.initials: F
authors.lastname: Afrin
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000009:adverse effects / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis; D031601:Azadirachta / Q000737:chemistry*; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D007155:Immunologic Factors / Q000009:adverse effects / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D007891:Leishmania / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D007896:Leishmaniasis / Q000188:drug therapy* / Q000469:parasitology; D008264:Macrophages / Q000469:parasitology; D008807:Mice, Inbred BALB C; D008853:Microscopy; D010936:Plant Extracts / Q000009:adverse effects / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D018515:Plant Leaves / Q000737:chemistry; D012639:Seeds / Q000737:chemistry; D018417:Th1 Cells / Q000276:immunology*; D016896:Treatment Outcome
chemical_list: D000871:Anthelmintics; D007155:Immunologic Factors; D010936:Plant Extracts