title: Topical application of serine proteases from Wrightia tinctoria R. Br. (Apocyanaceae) latex augments healing of experimentally induced excision wound in mice.
abstract: BACKGROUND
  Wrightia tinctoria R. Br. (Apocyanaceae) is a folk medicinal plant known to have immunomodulatory, anti-inflammatory and antihemorrhagic potential. Wrightia tinctoria latex is used for treatment of various clinical conditions including psoriasis, blisters, mouth ulcers, and extensively for topical application on fresh wounds to promote accelerated healing.
  
  
  OBJECTIVE
  To investigate the wound healing potential of Wrightia tinctoria latex proteases using a mouse model.
  
  
  METHODS
  Proteolytic activity of Wrightia tinctoria latex proteases (WTLP) was determined on various substrates (casein, gelatin and collagen (type-I and IV)). The thermal stability and the class of proteases present in WTLP were determined using heat treatment and specific protease inhibitors, respectively. Excision wound model in mice was used to evaluate the healing potential of WTLP application (twice daily, 10mg/kg). Neosporin, a standard drug, was used for comparison. The progression of healing was monitored using physical (wound contraction), biochemical (collagen content, catalase and MMP activity) and histological examinations.
  
  
  RESULTS
  WTLP contains thermostable serine proteases, which are completely inhibited by PMSF. WTLP showed strong caseinolytic, gelatinolytic and collagenolytic activity. The excision wound healing rate upon WTLP treatment was significantly higher than (>2-fold) the control group (49% vs. 18%, (**)p<0.01) on day 3 and throughout the study. PMSF pre-treated and heat denatured WTLP failed to promote wound healing. In addition, serial biochemical analysis of the granulation tissue demonstrated 1.5-fold more (2444 ± 100 vs. 1579 ± 121 µg/100mg tissue) hydroxyproline content and 5.6-fold higher catalase activity (16.7 ± 1.3 vs. 3 ± 0.3 units/mg) compared to controls. Further, the enhanced collagen content and matrix metalloproteinase activity correlated with wound contraction rate following WTLP and Neosporin treatment. Histological analysis on day 9 confirmed complete epithelialization, re-establishment of skin structure and accelerated wound healing following WTLP treatment.
  
  
  CONCLUSIONS
  The thermostable serine proteases of Wrightia tinctoria latex are directly involved in the wound healing process. Our findings provide a biochemical basis for the role of WTLP in the enhancement of wound healing. The study supports traditional topical application of Wrightia tinctoria latex on fresh wounds to promote accelerated healing.
authors.affiliation: Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India.
authors.forename: M
authors.initials: M
authors.lastname: Yariswamy
authors.forename: H V
authors.initials: HV
authors.lastname: Shivaprasad
authors.forename: Vikram
authors.initials: V
authors.lastname: Joshi
authors.forename: A N
authors.initials: AN
authors.lastname: Nanjaraj Urs
authors.forename: A
authors.initials: A
authors.lastname: Nataraju
authors.forename: B S
authors.initials: BS
authors.lastname: Vishwanath
mesh_terms: D000287:Administration, Topical; D000818:Animals; D029062:Apocynaceae / Q000737:chemistry* / Q000201:enzymology; D004195:Disease Models, Animal; D004355:Drug Stability; D004795:Enzyme Stability; D020834:Ethnopharmacology; D005260:Female; D006358:Hot Temperature; D007194:India; D007840:Latex; D008297:Male; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology; D057057:Serine Proteases / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D014945:Wound Healing / Q000187:drug effects*; D014950:Wounds, Penetrating / Q000188:drug therapy* / Q000473:pathology
chemical_list: D007840:Latex; D011480:Protease Inhibitors; D057057:Serine Proteases
keywords: BSA; Bovine serum albumin; ECM; EDTA; Ethylene diamine tetra-acetic acid; Extracellular matrix; IAA; Iodo acetic acid; Latex proteases; MMPs; Matrix metalloproteinases; PMSF; Para dimethyl amino benzaldehyde; Phenyl methyl sulphonyl fluoride; Protease inhibitors; SDS-PAGE; Serine proteases; Sodium dodecyl sulphate polyacrylamide gel electrophoresis; WTLP; Wound excision healing; Wrightia tinctoria; Wrightia tinctoria latex proteases; p-DMAB