| Proanthocynidins, a group of polyphenolics compounds, widely distribute in stem bark, leaves, flowers and fruits of various plants, possess a broad spectrum of biological and chemical activities. Therefore, proanthocynidins are a good source for the development of pharmaceutical products. Tyrosinase (EC1.14.18.1) is a copper containing redoxidase extensively distributed in nature. In human, tyrosinase is responsible for skin pigmentation abnormalities, such as flecks and defects. Recently, tyrosinase was reported to relate with Parkinson’s diseases and other neurodegenerative diseases. In addition, tyrosinase is responsible for anthopod cuticle sclerotization. Furthermore, tyrosinase causes browning in vegetables, fruits and mushrooms. Therefore, tyrosinase is very important in the fields of medicine and agriculture industry. Especially in the cosmetic industry, the development and screening of potent tyrosinase inhibitors are especially attractive.In this research, the extraction conditions of phenolics from Ficus virens leaves were optimized using response surface methodology (RSM). Extraction ability of phenolics (EAP) and flavonoids (EAF),2,2-diphenyl-l-pierylhydrazyl (DPPH) free-radical scavenging potential and the ferric reducing/antioxidant power (FRAP) were used as quality indicators. The results of single-factor experiments showed that temperature, ethanol concentration, extraction time and the number of extraction cycles were the main influencing variables. These provided key information for the central composite design. The results of RSM fitted well to a second degree polynomial model and more than98%of the variability was explained. The ideal extraction conditions for EAP, EAF, DPPH free-radical scavenging potential and FRAP were obtained. Considering the four quality indicators overall, the ideal extraction conditions determined were58%ethanol at57℃for37min with three extraction cycles. Linear correlations were also observed between EAP, EAF and antioxidant potential of the phenolics.The structure of the proanthocynidins from the leaves, fruit and stem bark of F. virens, from the fruit pericarp and fresh of Caryota ochlandra, and from the leaves of Polyalthia longifolia were characterized by13C nuclear magnetic resonance, high performance liquid chromatography electrospray ionization mass spectrometry coupled with thiolysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses. Both propelargonidin (PP) and procyanidin (PC) were found in all the proanthocynidins and B-type linkages were present in all the compounds (except the proanthocynidins from the leaves of P. longifolia), while epicatechin was found to be the monomer units of P. longifolia leaves proanthocynidins. The masses of the highest peaks among the proanthocynidins polymers with identical DP increased at a distance of288Da corresponding to the addition of one catechin/epicatechin monomer unit. The polymer chain lengths of the proanthocynidins from the leaves, fruit and stem bark were detected up to6-mer,12-mer, and15-mer, respectively. Those of the C. ochlandra fruit pericarp and fresh were both detected up to12-mers and those of P. longifolia leaves were detected up to14-mer. In addition to the predicted homopolyflavan-3-ol mass series mentioned above, each DP had several subsets of masses16Da lower in the spectra of proanthocynidins (except the proanthocynidins from the leaves of P. longifolia). These masses indicated that the polymer chains contained monomers with only one hydroxyl group (16Da) on the aromatic ring B. The proanthocynidins from C. ochlandra fruit fresh have a series of peaks increased at a distance of272Da which suggested that the oligomers consist of pure afzelechin/epi-afzelechin units.The proanthocynidins from the leaves, fruit and stem bark of F. virens, from the fruit pericarp and fresh of C. ochlandra, and from the leaves of P. longifolia were studied for their antioxidant activities through DPPH and ABTS free radical scavenging, and ferric reducing/antioxidant power (FRAP) assays. Among the different parts of F. virens, proanthocynidins from stem bark exhibited the most potent antioxidant activity; their IC5o value of DPPH and ABTS scavenging activities were109.20±0.99and80.82±3.31μg/mL, respectively; their FRAP values was688.34±24.14mg ascorbic acid equivalent/g dry weight (mg AAE/g d.w.). Proanthocynidins from fruit pericarp of C. ochlandra exhibited stronger antioxidant activity than those of fruit fresh. The IC50value of DPPH and ABTS free radical scavenging assays for C. ochlandra fruit pericarp and fresh were142.86±1.53and80.51±0.4μg/mL, and157.42±1.87and83.18±1.22μg/mL, respectively. The FRAP values for C. ochlandra fruit pericarp and fresh were373.09±5.02and317.91±13.06mg AAE/g d.w.. The IC50for DPPH·and ABTS·scavenging activity of proanthocynidins from P. longifolia leaves were89.32±12.07and76.79±5.88μg/mL, respectively. The FRAP value for proanthocynidins from P. longifolia leaves was710.54±142.82mg AAE/g d.w. The structure and antioxidant activity relationship analyses indicated that hydroxyl group at the proanthocynidins3position of B ring enhance their antioxidant capacity.The proanthocynidins from the leaves, fruit and stem bark of F. virens, from the fruit pericarp and fresh of C. ochlandra, and from the leaves of P. longifolia were studied for their effects on mushroom tyrosinase activities. Moreover, the inhibition mechanisms and kinetics of the proanthocynidins from the leaves, fruit and stem bark of F. virens were investigated. Furthermore, fluorescence quenching spectroscopy, copper interaction, and molecular docking were utilized to unravel the underlying inhibition mechanism of the proanthocynidins from the leaves, fruit and stem bark of F. virens on tyrosinase. The proanthocynidins from the leaves, fruit and stem bark of F. virens were potent and mix type tyrosinase inhibitors. The IC50values for the leaves, fruit and stem bark proanthocynidins on the inhibition of monophenolase activity were131.67,99.89, and106.22μg/mL, respectively. The lag period was shortened with increasing concentration of the leaves, fruit and stem bark proanthocynidins. The IC50values of diphenolase activity were estimated to be128.42±0.45,43.07±0.62, and74.27±0.68μg/mL for the proanthocynidins from the leaves, fruit and stem bark of F. virens, respectively. Inhibition mechanism study revealed that the proanthocynidins were able to quench and form copper interaction with the enzyme catalytic site. Additionally, o-quinone (the product of tyrosinase) could be scavenged by the proanthocynidins. Interestingly, the proanthocynidins from C. ochlandra fruit pericarp inhibited tyrosinase activity, however, fruit fresh proanthocynidins contained significant amount of catechin which could be oxidized by tyrosinase catalyzation. Lastly, the IC50for diphenolae activity of P. longifolia leaves proanthocynidins was773.09±1.47μg/mL.In summary, proanthocynidins, potent tyrosinase inhibitors, were purified in this study. Furthermore, their structure, antioxidant activities and tyrosinase inhibition mechanisms were investigated which would provided scientific foundations for their usage as antioxidant and whitening cosmetics. |
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