Preparation Of Mulberroside A In The Branch Bark Of Mulberry And Its Inhibition Mechanism To Tyrosinase

Mulberry is an abundant and renewable natural resource whose branches andstems however, have not been put to good use and are thus a wasted resource. Thisstudy was carried out to extract bioactive ingredients from an ethanol-water extract ofthe bark from the branches of cultivated mulberry Husang-32(Morus multicaulisPerr.). The extract was then fractioned over a D101macroporous resin column andeluted with a gradient elution of ethanolic-water. A main component in30%ethanolelution was further purified by semi-preparative HPLC-DAD and identified asmulberroside A by LC-MS,1H and13C NMR spectra.The content of mulberroside A in extracts from different parts of mulberry wasexamined by RP-HPLC. The results showed a significant difference in different partsof different mulberry species. The contents of mulberroside A in roots of Sha-2andHusang-32were about32-fold and2.4-fold higher than in their branch barks,respectively, but there was almost no difference between the root and branch bark ofYanbianqiuyu. The mulberroside A can be hardly detected.In order to understand the range of mulberroside A that might be found inmulberry, the content of mulberroside A from branch bark of100mulberry specieswas examined. The quantitative analysis results revealed a great diversity (about55-fold) in levels of mulberroside A in the different cultivated species of mulberry,varying between0.494and25.47g/mg, respectively. The Dazhongsang, Heiyousang,Muzhouqing and7311contained more mulberroside A, and the content was high at25.47,22.28,22.01and20.11g/mg, respectively. The content of mulberroside A inHuangzhisang, Sifangai, Sha-2and Tang-10was relatively low, and the content was0.61,0.84,0.49and0.47g/mg, respectively.Mulberroside A is an oxyresveratrol diglycopyranoside where the4-OH and3′-OH have been substituted by two glucoses, so we studied the effects of mulberroside A, compared with oxyresveratrol, on the generation process ofdopachrome. The results suggested that mulberroside A was a real tyrosinase inhibitor,which can inhibit the generation of dopachrome by affecting the activities ofmonophenol oxidase and diphenol oxidase of tyrosinase in vitro.The effects of mulberroside A on the activities of monophenol oxidase anddiphenol oxidase of tyrosinase were studied, and the results showed that mulberrosideA had a stronger inhibitory effects on the monophenol oxidase than on the diphenoloxidase, and its IC50was0.13mol/L and7.35mol/L, respectively.Meanwhile, the kinetic analysis of mulberroside A and oxyresveratrol onmonophenol oxidase and diphenol oxidase of tyrosinase was carried out. For themonophenol oxidase activity, the inhibition of mulberroside A and oxyresveratrol onthe monophenol oxidase of the enzyme was reversible and belonged to mix-type1,and the inhibition constants KIwere determined to be29.22mol/L and92.60mol/L,and KISwere determined to be37.70mol/L and66.20mol/L, respectively. For thediphenol oxidase activity, there was some difference between mulberroside A andoxyresveratrol, the mulberroside A belonged to competitive inhibition and KIweredetermined to be1.56mol/L, but oxyresveratrol belonged to noncompetitiveinhibition and KI=KIS=2.95mol/L.In this study, the efficient method of preparation of mulberroside A frommulberry branch bark was builded, and the content of mulberroside A from thedifferent part of mulberry and branch barks of different mulberry species, wasanalysised. Meanwhile, the inhibitory activity on tyrosinase of mulberroside A wasexamined. So, it has significant meaning for development and utilization of mulberryand mulberroside A to study on this subject.