| Tyrosinase is the rate-limiting enzyme in melanin synthesis, so tyrosinase inhibitors can beused in the treatment of pigmentation diseases, food preservation and bio-pesticides. In recentyears, the development and research of a safe, specific, effective and long-term use naturaltyrosinase inhibitors has become focus and direction for R&D institutions and drug companieswith extensive use of tyrosinase inhibitors. However, Natural tyrosinase inhibitors is easilyoxidized, and its content is very low, it can be lost or loss of activity easily in the separation andpurification process, and it is not easy to obtain a highly active monomers in the conventionalrefining process. Lavender flowers was used as material in the study, a new screening methods ofnatural tyrosinase inhibitor based on HPLC post-column derivatization was establishedsuccessfully, and the active target compounds were isolated, it is for the dewelopment of noveldrugs of tyrosinase inhibitor, food preservation agents and whitening cosmetic ingredient to laythe foundation. Meanwhile, the constituents of lavender flowers was studied incompletely, itschemical composition was systematically separated in the futher. The main contents and resultsare as follows:1. Inhibitory effects of petroleum ether extracts, chloroform extracts, ethyl acetate extracts,n-butanol extracts and water extracts of lavender flowers on tyrosinase were studied. The resultsshowed that the effective inhibitory components of lavender flowers was mainly in water extracts,its IC50was0.96mg/mL, kinetic studies showed that the inhibitory effect is reversible, inhibitorytype is linear mixed inhibition, the inhibition constants of the free enzyme and enzyme-substratecomplexes were3.00and12.00mg/mL, respectively.2. The impacts of temperature, pH, reaction time, substrate concentration, enzymeconcentration, organic solvent system, co-solvents and metal ions on the activity of tyrosinase inthe offline conditions were discussed. Results showed as follows:2mmol/L L-DOPA,20mmol/L PBS（pH6.8）,30oC maintaining10min,150μg/mL tyrosinase, the reaction time was6min；40%methanol or30%acetonitrile can be tolerated at about40%remaining activity of tyrosinase;cosolvents or metal ion was no significant promotion on the activity of tyrosinase in an organicsolvent system.3. A screening model of online natural tyrosinase inhibitor based on HPLC post-columnderivatization was established, kojic acid was used as test sample, the model was optimized, theparameters were determined: the flow rate of L-DOPA solution and tyrosinase solution are0.6mL/min, their concentration were1mmol/L and62.5μg/mL, respectively; flow rate of carrier(20mmol/L pH6.8PBS) was0.3mL/min; the length of reaction tube was20m, the reaction timewas1.619min; oven temperature was30℃; wavelength was475nm. Four known potent tyrosinaseinhibitors were used as sample for reliability verification of model, and the different extracts oflavender flowers were screened with the new model, one tyrosinase inhibitory active substancefrom water extracts was screened, it was identified as5-hydroxymethyl furfural (1) by nuclearmagnetic resonance and comprehensive analysis, it was isolated from the lavender at the first time.4. A rapid, sensitive and highly accurate flow injection-spectrophotometric assay fordetermining the impact of natural products on the activity of tyrosinase was established, thevolume of loop is20μL, other parameters are identical to newly-created online screening model.5. Five compounds from water extract and n-butanol extract of lavender flowers wereisolated with utilization of a variety of separation means like silica gel column, low pressurepreparative liquid chromatography and preparative liquid chromatography and Spectroscopicidentification methods, they were melilotoides (2), Danshensu (3), n-butyl3,4dihydroxyphenylla-ctate(4), rosemary acid (5) and Dichotomoside E (6), respectively. Compounds2-4are isolatedfrom lavender at the first time. |
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