title: Improved TLC Bioautographic Assay for Qualitative and Quantitative Estimation of Tyrosinase Inhibitors in Natural Products.
abstract: BACKGROUND
  TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays.
  
  
  OBJECTIVE
  An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study.
  
  
  METHODS
  L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent.
  
  
  RESULTS
  The limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-β-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay.
  
  
  CONCLUSIONS
  Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Key Laboratory of Standardisation of Chinese Medicines of Ministry of Education, The Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.|Shanghai TCM-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200082, China.
authors.forename: Jinge
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Shanghai Edible Fungi Institute, Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
authors.forename: Qingjiu
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Key Laboratory of Standardisation of Chinese Medicines of Ministry of Education, The Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wu
authors.affiliation: Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, 201203, China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Cheng
mesh_terms: D000818:Animals; D001688:Biological Products / Q000737:chemistry*; D045744:Cell Line, Tumor; D002855:Chromatography, Thin Layer / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results
chemical_list: D001688:Biological Products; D004791:Enzyme Inhibitors; D014442:Monophenol Monooxygenase
keywords: Rhodiola sacra; TLC bioautography; kojic acid; tyrosinase inhibitor