title: Exploration of anti-Malassezia potential of Nyctanthes arbor-tristis L. and their application to combat the infection caused by Mala s1 a novel allergen.
abstract: BACKGROUND
  Malassezia commensal yeasts along with multitude of antigens have been found to be associated with various skin disorders including Pityriasis versicolor (PV). Amongst them Mala s1, a 37 kDa protein has been proved to be a major allergen reacting with a large panel of sera. However, there exists no therapeutic alternative to combat such problems in form of plant based natural compounds. The purpose of this study is in the first place, to determine the anti-Malassezia activity of Nyctanthes arbor-tristis L. (NAT) ethanolic leaf extract through turbidimetric growth curves, disruption of plasma membrane and secondly, it aims to present in silico validation of its active constituents over Mala s1a novel allergen.
  
  
  METHODS
  The antifungal susceptibility 50 % ethanolic extract of NAT was determined by broth microdilution method according to CLSI guidelines. Further MICs and IC50 were determined spectrophotometrically using the software SoftMax® Pro-5 (Molecular Devices, USA). Active constituents mediated disruption of plasma membrane was studied through flowcytometry by permeabilization of fluorescent dye Propidium Iodide (PI). Antioxidant activity of the extract was determined using the DPPH stable radical. Molecular validation of fungal DNA from the extract was observed using PCR amplification. In silico analysis of its active constituents over Mala s1 was performed using HEX software and visualized through Pymol.
  
  
  RESULTS
  The anti-Malassezia potential of NAT leaf extracts reflected moderate MIC 1.05 μg/μl against M. globosa, while least effective against M. restricta with MIC 1.47 μg/μl. A linear correlation coefficient R (2) = 0.866 was obtained in case of M. globosa while minimum was observed in M. restricta with R (2) = 0.732. The flow cytometric data reveal ~ 75 % cell death when treated with active constituents β-Sitosterol and Calceolarioside A. The docking confirmations and the interaction energies between Mala s1 and the active constituents (β-Sitosterol and Calceolarioside A) from extracts showed an effective binding which suggests Mala s1 as efficient allergen for site specific targeting.
  
  
  CONCLUSIONS
  This study revealed that Nyctanthes arbor-tristis L. (NAT) extracts possess high anti-Malassezia potential which is driven mainly by disruption of plasma membrane. Also in silico validation and molecular modeling studies establishes Mala s1 as a novel allergen that could be a potential target in disease treatment. Our results would also provide a foundation for the development of new therapeutic approach using NAT extract as lead compound with high antioxidant property as an added trait for skin care.
authors.affiliation: Centre for Medical Diagnostic and Research (CMDR), Motilal Nehru National Institute of Technology (MNNIT), Allahabad, 211004, India. rohit_ernet@yahoo.co.in.
authors.forename: Rohit K
authors.initials: RK
authors.lastname: Mishra
authors.affiliation: Nanotechnology Application Centre (NAC), University of Allahabad, Allahabad, 211002, India. vanimish@gmail.com.
authors.forename: Vani
authors.initials: V
authors.lastname: Mishra
authors.affiliation: Department of Botany, Biological Product Lab., University of Allahabad, Allahabad, 211002, India.
authors.forename: Anand
authors.initials: A
authors.lastname: Pandey
authors.affiliation: Department of Botany, Biological Product Lab., University of Allahabad, Allahabad, 211002, India.
authors.forename: Amit K
authors.initials: AK
authors.lastname: Tiwari
authors.affiliation: Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture Technology and Sciences, Allahabad, 211007, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Pandey
authors.affiliation: Centre for Medical Diagnostic and Research (CMDR), Motilal Nehru National Institute of Technology (MNNIT), Allahabad, 211004, India.
authors.forename: Shivesh
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Nanotechnology Application Centre (NAC), University of Allahabad, Allahabad, 211002, India.
authors.forename: Avinash C
authors.initials: AC
authors.lastname: Pandey
authors.affiliation: Department of Botany, Biological Product Lab., University of Allahabad, Allahabad, 211002, India. anupambplau@rediffmail.com.
authors.forename: Anupam
authors.initials: A
authors.lastname: Dikshit
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000378:metabolism; D000935:Antifungal Agents / Q000378:metabolism / Q000627:therapeutic use*; D000946:Antigens, Fungal / Q000276:immunology* / Q000378:metabolism; D000975:Antioxidants / Q000627:therapeutic use; D002109:Caffeic Acids / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D003881:Dermatomycoses / Q000188:drug therapy*; D005960:Glucosides / Q000378:metabolism; D007194:India; D008290:Malassezia / Q000276:immunology*; D008826:Microbial Sensitivity Tests; D029592:Oleaceae / Q000737:chemistry*; D010936:Plant Extracts / Q000378:metabolism / Q000627:therapeutic use*; D012855:Sitosterols / Q000378:metabolism
chemical_list: D000485:Allergens; D000935:Antifungal Agents; D000946:Antigens, Fungal; D000975:Antioxidants; D002109:Caffeic Acids; D005960:Glucosides; C510167:Mala s 1 allergen, Malassezia sympodialis; D010936:Plant Extracts; D012855:Sitosterols; C025473:gamma-sitosterol; C083066:calceolarioside A
keywords: AUGC; Anti-Malassezia susceptibility; Flow cytometry; Mala s1; Molecular docking; Nyctanthes arbor-tristis L. (NAT)