Study On Tyrosinase Inhibitors From Mulberry (Morus Alba L.) Leaves

Mulberry(Morus alba L.)leaves were collected from a commercial orchard in Huzhou,Zhejiang Province and then employed to select and identifiy tyrosinase inhibitors.The extraction efficiency of mulberry flavonoids was evaluated.Furthermore,the effect of mulberry flavonoids on tyrosinase activity in relation to their antioxidant abilities and antibacterial activities were investigated.The major results obtained in this study were as follows.1.The inhibitory extent of tyrosinase activity by mulberry leaves followed the harvest seasons,i.e.autumn＞summer＞spring.Mulberry leaves collected in autumn was extracted with 50%ethanolic,and then purified using different polar solvents,polyamide column chromatography,silica gel chromatography and recrystallization.The final extraction yield of the tyrosinase inhibitors from mulberry leaves was determined to be 0.025 mg/g.The major compounds of the mulberry extract were preliminarily identified as flavonoids.The inhibition of tyrosinase activity by mulberry extract exhibited a reversible effect. 2.The highest extraction yield of flavonoids from mulberry leaves was obtained by ultrasound assisted-extraction,followed by ultra high pressure extraction,microwave assisted-extraction,enzymatic assisted-extraction,alkali-solution plus acid-isolation and soxhlet extraction.The optimum ultrasound-assisted extraction conditions of flavonoids from mulberry leaves were determined to be 70%ethanol(solvent),1:20 (material to solvent solution),200 W(ultrasound power)and 10 min (ultrasound time),following subsequent an extraction time of 1.5 h at 70℃,with an final extraction yield of 28.8 mg/g.Five flavonoids were purified and then identified as Rutin,Isoquercitrin,Astragalin,Morin and Kaempferol from the mulberry extract.3.Mulberry flavonoids exhibited a inhibitory effect on tyrosinase activity.The inhibitory effect of mulberry flavonoids on monophenolase activity was stronger than diophenolase activity of tyosinase.The 50% inhibition concentrations(IC₅₀)to monophenolase and diophenolase activities of tyosinase were 200 and 300μg/mL.It may be due to that the conformation of monophenolase was easier to change than that of diophenolase in the active sites of tyrosinase and a requirement of monophenolase for geometry and electronic effect was stricter than that of diphenolase,resulting in a decrease in the stability of the monophenolase.Furthermore,various flavonoid monomers showed a interaction effect on tyrosinase activity.4.The inhibition of the mixture of various mulberry flavonoid monomers on tyrosinase activity was reversible.The effects by combined flavonoid monomers did not result in a permenant change of conformation of tyrosinase but just cause the inhibition in the enzymatic activity.According to the flavonoid structure,the inhibitory effects of mulberry flavonoids on tyrosinase activity may be due to the complexation of OH present in the flavonoids and Cu²⁺present in the active sites of tyrosinase,or/and due to the similar structure of the flavonoids to the enzyme substrate tyrosinase.The mixture of various flavonoid monomers reduced the steady-state of and extended the lag time of monophenolase activity of tyosinase. While the mixture of various flavonoid monomers exhibited a mixed-type effect on diphenolase activity of tyosinase.The inhibition constants for the inhibitor binding with free enzyme(E),KI and enzyme-substrate(ES) complex,KIS were estimated to be 176.01 and 259.20μg/mL, respectively.5.Flavonoids present in mulberry leaf exhibited a strong antioxidant ability,which were related to their inhibition of tyrosinase activity.6.Mulberry flavonoids had a distinct antimicrobial activity.The minimal inhibitory concentrations(MIC)of mulberry flavonoids against Staphylococcus.aureus,Escherichia coli and Bacillus subtilis were 0.0125,0.025 and 0.05 mg/mL,respectively.