Avocado Proanthocyanidins as a Source of Tyrosinase Inhibitors

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Avocado Proanthocyanidins as a Source of Tyrosinase Inhibitors: Structure Characterization, Inhibitory Activity, and Mechanism Wei-Ming Chai,* Man-Kun Wei, Rui Wang, Rong-Gen Deng, Zheng-Rong Zou, and Yi-Yuan Peng* College of Life Science, and Key Laboratory of Small Functional Organic Molecule, Ministry of Education, Jiangxi Normal University, Nanchang, Jiangxi 330022, People’s Republic of China S Supporting Information *

ABSTRACT: Proanthocyanidins were purified from avocado (Persea americana) fruit, and their structures were analyzed by matrix-assisted laser desorption/ionization−time-of-flight mass spectrometry (MALDI−TOF MS) and high-performance liquid chromatography−electrospray ionization−QTRAP mass spectrometry (HPLC−ESI−QTRAP MS) techniques. The results obtained from mass spectrometry (MS) analysis demonstrated that the proanthocyanidins were homo- and heteropolymers of procyanidins, prodelphinidins, propelargonidins, and procyanidin gallate. From the enzyme analysis, the results showed that they could inhibit the monophenolase and diphenolase activities of tyrosinase. The inhibition mechanism of the proanthocyanidins on the enzyme was further studied, and the results indicated that they were reversible and competitive inhibitors. Finally, the results acquired from molecular docking, fluorescence quenching, and copper ion interacting tests revealed that adjacent hydroxyl groups on the B ring of proanthocyanidins could chelate the dicopper catalytic center of the enzyme. In addtion, proanthocyanidins were proven to be an efficient quencher of substrates. This study would lay a scientific foundation for their use in agriculture, food, and nutrition industries. KEYWORDS: avocado, proanthocyanidins, structure, tyrosinase inhibitors, inhibitory mechanism, molecular docking

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INTRODUCTION Tyrosinase, also named polyphenol oxidase, is a multifunctional copper-containing oxidase found in a broad variety of organisms. It catalyzes both a monophenolase reaction cycle (monophenolase activity) and a diphenolase reaction cycle (diphenolase activity) of melanin synthesis (part 1 of Figure 1).1 The enzyme plays critical roles in the browning of food,2 development of insects,3 and melanogenesis.1 This indicates that tyrosinase inhibitors are of great significance in the fields of agriculture, food, and cosmetics. In our previous study, proanthocyanidins extracted from kiwifruit pericarp showed potent tyrosinase inhibitory activity.4 Proanthocyanidins are a class of polyphenols widely distributed in cereals and legume seeds and particularly abundant in some fruits. Owing to their biological activities, proanthocyanidins have aroused enough interests in the areas of food, agriculture, and nutrition. It is reported that these compounds possess many physiological effects by acting as antioxidant, antiviral, anticarcinogen, cardiopreventive, antibacterial, and neuroprotective agents.5 However, it is universally acknowledged that the bioactivity capacity of plant proanthocyanidins largely depends upon their structure, especially the molecular weight (degree of polymerization).6,7 Therefore, it is necessary to study the structures of proanthocyanidins. Proanthocyanidins are polymers of flavan-3-ols, which present a wide variety of chemical structures (part 2 of Figure 1). The structures of proanthocyanidins vary based on the diversity of the monomer units, interflavan linkage, degree of polymerization, and substituent of the 3-hydroxyl group.8 Because of the complexity and diversity, the characterizations of structures of proanthocyanidins remain difficult. In this study, matrixassisted laser desorption/ionization−time-of-flight mass spec© XXXX American Chemical Society

trometry (MALDI−TOF MS) and high-performance liquid chromatography−electrospray ionization−QTRAP mass spectrometry (HPLC−ESI−QTRAP MS) methods were employed to characterize the structures of proanthocyanidins. Avocado (Persea americana) is an important commercial fruit tree, which can be found in most tropical and subtropical areas currently. Avocado fruits contain lots of unsaturated fatty acids, vitamins B and E, carotenes, fibers, and other nutrients.9 Prior authors have focused on antioxidant and antifungal activities of extracts obtained from different parts of avocado.10−12 However, to the best of our knowledge, there is no knowledge about the structure, antityrosinase activity, and mechanism of avocado fruit proanthocyanidins. Exploiting the phytochemical content of avocado may create new products and add value to the avocado industry. Therefore, the research objective is to determine the structures as well as antityrosinase activities of proanthocyanidins extracted from avocado fruit. This work will be conducive to the development and design of natural preservative, insecticide, and skin-whitening agents.

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MATERIALS AND METHODS

Chemicals and Materials. All analytical-grade solvents, including acetone, petroleum ether, ethyl acetate, and methanol, were purchased from Sinopharm (Shanghai, China). High-performance liquid chromatography (HPLC)-grade acetonitrile was also purchased from Sinopharm. L-Tyrosine, 3,4-dihydroxyphenylalanine, mushroom tyrosinase, Sephadex LH-20, benzyl mercaptan, trifluoroacetic acid, Received: June 23, 2015 Revised: August 9, 2015 Accepted: August 10, 2015

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DOI: 10.1021/acs.jafc.5b03099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of proanthocyanidins, flavan-3-ol monomer units, and substrates of tyrosinase. (1) Processes of melanogensis and structural similarity between epicatechin/catechin and L-DOPA. (2) Chemical structures of flavan-3-ol monomer units and proanthocyanidins. Reversed-Phase HPLC−ESI−QTRAP MS Analysis after Thiolysis Reaction. Reversed-phase HPLC−ESI−QTRAP MS analysis was completed in the method described by Zhou et al. 7 Proanthocyanidins were thiolysis-degraded in the presence of benzyl mercaptan, and then the degradation products were injected into an Agilent 1200 system (Agilent, Santa Clara, CA) interfaced to a QTRAP 3200 (Applied Biosystems, Foster City, CA) with a 250 × 4.6 mm inner diameter, 5.0 mm Hypersil ODS column (Elite, Dalian, China). The mean degree of polymerization (mDP) was calculated on the basis of the following equation:

Amberlite IRP-64 cation-exchange resin, cesium chloride, 2,5dihydroxybenzoic acid, and all HPLC standards were purchased from Sigma-Aldrich (St. Louis, MO). Arbutin was obtained from Aladdin (Shanghai, China). The fresh fruits of avocado were picked from the trees growing in Xiamen University (Xiamen, China) in August 2012. They were immediately washed, freeze-dried, ground, and stored at −20 °C before further processes. Extraction and Purification of Avocado Proanthocyanidins. Powders (10.0 g) of the avocado fruit were ultrasonically extracted 3 times with acetone/water (70:30, v/v, each 200 mL) solution at 25 °C. Simultaneously, 0.1% ascorbic acid was added to prevent the oxidation of proanthocyanidins. Petroleum ether and ethyl acetate were then selected as an extractant to eliminate chlorophyll, lipophilic compounds, and low-molecular-weight phenolics. The remaining aqueous fraction was chromatographed on a Sephadex LH-20 column (50 × 1.5 cm inner diameter), which was eluted successively with 50% methanol/water and 70% acetone/water. The fraction of 70% acetone/water was retained; acetone was eliminated; and then the aqueous fraction freeze-dried to obtain purified proanthocyanidins. MALDI−TOF MS Analysis. The MALDI−TOF MS spectra were obtained through a Bruker Reflex III (Germany). The experiment was completed according to the conditions and steps previously described.4 As the irradiation source, a pulsed nitrogen laser (337 nm) would last 3 ns. Cs+ and 2,5-dihydroxybenzoic acid were selected as the cationization and matrix, respectively. The sample solutions (10 mg/mL), the cesium chloride solution (1.52 mg/mL), and the matrix solution (10 mg/mL) were mixed at a volumetric ratio of 1:1:3.

mDP = (total area of the extender units) /(total area of the terminal units) + 1 Enzyme Assay. L-Tyrosine was selected as a substrate of monophenolase, and 3,4-dihydroxyphenylalanine was used as a substrate of diphenolase. The final concentrations of the enzyme were 16.67 and 3.33 mg/mL for the monophenolase and diphenolase analyses, respectively. The experiment was repeated 3 times. Their results were expressed in IC50 value (μg/mL). The inhibition type depended upon the Lineweaver−Burk plot. Arbutin was selected as a positive control. The procedure and conditions for the enzyme reaction were carried out as described in our previous study.4 Fluorescence Quenching Analysis. The fluorescence test of tyrosinase and proanthocyanidins was performed on a fluorescence spectrophotometer (Varian Cary Eclipse). Sample solution, tyrosinase solution, and sodium phosphate buffer (pH 6.8) were mixed at a B


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Journal of Agricultural and Food Chemistry volumetric ratio of 1:2:7 (v/v/v). The final reaction total volume was 3 mL. The analysis was carried out with an excitation wavelength of 290 nm at 25 °C. The Stern−Volmer equation describes fluorescence quenching13 F0/F = 1 + Kqτ0[Q ] = 1 + KSV[Q ] where F0 and F are the fluorescence intensities in the presence and absence of the quencher, respectively, Kq is the bimolecular quenching constant, τ0 (10−8 s) is the lifetime of the fluorophore in the absence of the quencher, [Q] is the concentration of the quencher, and KSV is the Stern−Volmer quenching constant. There exist two types of fluorescence quenching mechanisms: static and dynamic.14 When the type was static, the apparent binding constant (KA) and the binding affinity (n) are estimated by the following equation:15 log[(F0 − F )/F ] = log KA + n log[Q ] The fluorescence assays of L-tyrosine/3,4-dihydroxyphenylalanine and proanthocyanidins were also performed on a fluorescence spectrophotometer (Varian Cary Eclipse). In brief, L-tyrosine (12 mM) or 3,4dihydroxyphenylalanine (0.5 mM) solution, sample solution, and sodium phosphate buffer (pH 6.8) were mixed at a volumetric ratio of 1:1:3 (v/v/v) at 25 °C. The mixture was preincubated for 30 s before fluorescence spectra analysis. The excitation wavelength was 290 nm. The experiment was repeated 3 times. Determination of Cu2+ Chelating Activity. A total of 100 μL of copper sulfate solution with different concentrations (25, 50, and 100 μM) and 100 μL of sample (1 mg/mL) solution were added to sodium phosphate buffer (50 mM, pH 6.8). The final volume was 1 mL, and the mixture was then shaken well and kept at 25 °C for 1 min prior to ultraviolet−visible (UV−vis) and fluorescence quenching analyses. Molecular Docking of Tyrosinase with Ligands. Protein− ligand docking was completed by Molecular Operation Environment (MOE) 2010 software. In present study, we selected the structure of the oxy-tyrosinase of Streptomyces castaneoglobisporus as the initial model for docking simulations. ChemBioDraw Utra 12.0 was used to prepare the three-dimensional (3D) structures of the ligands. The structure models of the protein and ligands were energy-minimized before docking. Then, hydrogens were added to the protein and ligands. The refinement module of molecular docking was set to force field and retains scoring set to 10. The docking poses ranged according to the MM/GBVI binding free energy scoring. The binding mode was judged by the docked conformation, which had the lowest energy value. Statistical Analysis. SPSS 19.0 was applied to the calculations. Results of antityrosinase analyses were indicated as the mean ± standard deviation.

Figure 2. MALDI−TOF positive-ion (Cs+) mode mass spectra of avocado proanthocyanidins in the (A) reflectron mode and (B) linear mode. The y axis represents absolute intensity (ai), that is, the number of ions of each species that reach the detector.

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RESULTS AND DISCUSSION MALDI−TOF MS Analysis. The MALDI−TOF MS technique is an effective method for the characterization of proanthocyanidins in food science.4,7,16,17 It has the potency to distinguish molecular weight differences because of the hydroxylation pattern in the B ring (Δ 16 Da), 3-O-galloylation (Δ 152 Da), or A-type linkage (Δ 2 Da).16 Figure 2A displayed the MALDI−TOF mass spectra of avocado fruit proanthocyanidins, recorded as Cs+ adducts in the reflection mode. Peaks increased equably from 999.00 Da (3-mers) to the 6183.42 Da (21-mers) at a distance of 288 Da, which was in accordance with the addition of one (epi)catechin. Therefore, it could be concluded that the main constitute unit is (epi)catechin. For each multiple, masses of 16 Da higher and lower were also detected in spectra when they were enlarged (Figure 2A). These masses were heteropolymers of repeating constitution units, which indicated the presence of (epi)gallocatechin and (epi)afzelechin. These results further demonstrated the coexistence of procyanidins, prodelphinidins, and propelargo-

Figure 3. Reversed-phase HPLC−ESI−QTRAP MS chromatograms of proanthocyanidins from avocado after thiolytic degradation.

nidins in avocado fruit proanthocyanidins, which were dominated by procyanidins. However, in comparison to procyanidins, there were few prodelphinidins and propelargonidins. Besides the series of peaks described above, mass signals following the main groups of peaks at a distance of 152 and 132 Da were detected (Figure 2A). The 152 Da mass distance was in accordance with the addition of one galloyl group at the heterocyclic C ring.18 The 132 Da mass distance may be produced by the substitution of a pentoside18 or synchronous attachment of two Cs+ and the absence of a proton [M + 2Cs+ − H]+.19 Specifically, mass signals following the main groups of C


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Table 1. Effects of Avocado Proanthocyanidins on the Activity of Mushroom Tyrosinase for the Oxidation of L-Tyr and LDOPAa monophenolase

diphenolase

sample

IC50 (μg/mL)

IC50 (μg/mL)

inhibition

inhibition type

proanthocyanidins arbutin

40 ± 1.2 not detected

19.5 ± 0.6 a 1442.9 ± 2.77 b

reversible reversible

competitive competitive

a Values are expressed as the mean of triplicate determinations ± standard deviation. Different letters in the same column show significant differences from each other at the p < 0.05 level.

peak detected was epicatechin benzylthioether, while the other five smaller peaks were catechin, epicatechin, (epi)gallocatechin benzylthioether, catechin benzylthioether, and (epi)afzelechin benzylthioether. These results indicated that avocado fruit proanthocyanidins were mainly constituted of procyanidins, which were composed of an epicatechin unit. These results agreed well with the findings completed by the MALDI−TOF MS method. Besides, mDP of the proanthocyanidin polymers was calculated to be 12.3 ± 0.8. In fact, depolymerization by phenolysis (phloroglucinol) was also an efficient approach to determine the mDP of proanthocyanidins. Proanthocyanidin oligomers of 15 strawberry cultivars were depolymerized by phenolysis, and their mDP (3.4−5.8) was estimated.22 Effects of Avocado Fruit Proanthocyanidins on Monophenolase Activity. Effects of avocado fruit proanthocyanidins on monophenolase activity, where L-tyrosine was catalyzed by monophenolase, were first studied (see Figure 1-I of the Supporting Information). When the monophenolase activity was tested, a lag period (lag time) was observed. After the lag time, the monophenolase activity reached a steady-state rate, which was estimated by extrapolation of the linear portion of the product accumulation curve to the abscissa. Increasing concentrations of the avocado fruit proanthocyanidins not only extended the lag time (see Figure 1-III of the Supporting Information) but also decreased obviously the steady-state rate (see Figure 1-II of the Supporting Information). IC50 of proanthocyanidins on monophenolase activity was calculated as 40 ± 1.2 μg/mL (Table 1). Arbutin had little effect on the monophenolase activity (Table 1). The lag time was extended by 36 ± 0.8 s when the concentration of the proanthocyanidins was increased to 80 μg/mL. These results showed that avocado fruit proanthocyanidins were efficient inhibitors of monophenolase. Effect of Avocado Fruit Proanthocyanidins on Diphenolase Activity. Effects of avocado fruit proanthocyanidins on oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by diphenolase were investigated. The results showed that diphenolase activity was obviously reduced with increasing the concentration of the inhibitor, but it was not completely suppressed (see Figure 2-I of the Supporting Information). IC50 of proanthocyanidins on diphenolase activity was estimated to be 19.5 ± 0.6 μg/mL (Table 1). IC50 of arbutin was 1442.9 ± 2.77 μg/mL (Table 1). The molecular weight of proanthocyanidins was markedly larger than that of arbutin, and avocado fruit proanthocyanidins might therefore be considered as excellent tyrosinase inhibitors.

Figure 4. Changes in intrinsic tyrosinase fluorescence at different concentrations of avocado pulp proanthocyanidins. (A) Emission spectra 0, 1, 2, 3, and 4 of tyrosinase in the presence of proanthocyanidins are 0, 5, 10, 30, and 50 μg/mL, respectively. (B) Maximum florescence intensity changes. (C) Stern−Volmer plot describes the tyrosinase quenching caused by association with the quencher. (D) Plot of log[(F0 − F)/F] against log[Q] for tyrosinase and various concentrations of the quencher. F0 and F are the fluorescence intensities before and after the addition of the quencher.

peaks at a distance of 120 Da were also found in the mass spectrum. Additionally, the degree of polymerization of these compounds varied from 3-mers to 21-mers under the reflection mode, with the most intensity in 6-mers. However, the linear MALDI−TOF MS technique allowed for the test of avocado fruit proanthocyanidins up to 36-mers (Figure 2B). These results revealed structural heterogeneity and complexity of avocado fruit proanthocyanidins. The structures of proanthocyanidins extracted from avocado fruit were therefore successfully analyzed by employing MALDI−TOF MS for the first time. Analysis of Proanthocyanidins by Reversed-Phase HPLC−ESI−QTRAP MS after Thiolysis Reaction. Avocado fruit proanthocyanidins were depolymerized by thiolysis reaction, which was proven to be a powerful method.16,17,20 Benzylmercaptan was selected as the nucleophile. The result of the thiolysis reaction showed that terminal units were released as free monomers, but extender units were released as benzylthioether adducts.21 Figure 3 showed that the dominant

Table 2. Stern−Volmer Equation for the Interaction between Quenchers and Tyrosinase quencher proanthocyanidins

type of quenching static

KSV (μg/mL)−1

Kq (μg mL−1 s−1)−1

−3

(8 ± 0.2) × 10

(8 ± 0.2) × 10

5

D

KA (μg/mL)−1 (5 ± 0.4) × 10

−3

n 1.17 ± 0.02


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Figure 5. (1) Interaction of Cu2+ and proanthocyanidins. (2) Binding mode found from the ligand with tyrosinase residues using the lowest energy docked method. The receptor exposure differences were shown by the size and intensity of the turquoise discs surrounding the residues. The red arrows indicated the interaction of the ligand and the copper iron. Panels A, B, and C represented L-DOPA, epicatechin, and procyanidin dimer, respectively.

The inhibition mechanism of the avocado fruit proanthocyanidins on diphenolase activity was studied. It was revealed in Figure 2-II of the Supporting Information that, with the different concentrations of proanthocyanidins, the enzyme activity relied heavily on its concentration. The plots of the residual enzyme activity versus the concentrations of enzyme at different concentrations of proanthocyanidins gave a family of straight lines, which passed through the origin, indicating that the inhibition of the enzyme by proanthocyanidins was

reversible. With the proanthocyanidin concentrations increased gradually, the slopes of the lines were descending. The kinetic behavior of diphenolase in the process of oxidation of L-DOPA was then studied. The oxidation of LDOPA catalyzed by diphenolase followed Michaelis−Menten kinetics. Plot of 1/v versus 1/[S] gave a family of lines with a fixed intercept on the y axis but with different slopes. These lines intersected on the y axis, which indicated that avocado E


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± 0.4) × 10−3 (μg/mL)−1 and 1.17 ± 0.02, respectively (Table 2). Interaction of Cu2+ and Proanthocyanidins. Interaction between proanthocyanidins and Cu2+ were investigated using fluorescence analysis. The intrinsic fluorescence intensity of avocado fruit proanthocyanidins increased with the addition of Cu2+ (part 1 of Figure 5). The Cu2+ concentrations for curves 0, 1, 2, and 3 were 0, 25, 50, and 100 μM, respectively. The results revealed the binding of Cu2+ and proanthocyanidins. Molecular Docking. To better understand the mechanism of the potent antityrosinase activities of the proanthocyanidins, molecular docking were carried out using MOE. Interactions of the ligands (L-DOPA, epicatechin, and procyanidin dimer) and the virtual enzyme catalytic center were simulated. The results revealed that L-DOPA (panel A in part 2 of Figure 5), (epi)catechin (panel B in part 2 of Figure 5), and proanthocyanidin dimer (panel C in part 2 of Figure 5) could chelate with copper irons of tyrosinase. The results obtained from docking simulations were a good supplement of the interaction experiment between Cu2+ and proanthocyanidins. The results revealed that the copper ion chelating ability of proanthocyanidins with an adjacent hydroxyl group on the B ring may be a significant mechanism to explain their inhibitory potency on the tyrosinase. Fluorescence Quenching Analysis between the Substrate of Tyrosinase and Avocado Fruit Proanthocyanidins. The interaction between the proanthocyanidins and substrate of tyrosinase was investigated using the fluorescence quenching method. The result showed that intrinsic fluorescence intensity of L-tyrosine and 3,4-dihydroxyphenylalanine decreased distinctly with the addition of proanthocyanidins (Figure 6). The fluorescence intensity of L-tyrosine was reduced by 56.8% when the proanthocyanidin concentration reached 80 μg/mL (curve 4 in Figure 6A). Concentrations of curves 0, 1, 2, 3, and 4 were 0, 20, 30, 40, and 80 μg/mL, respectively (Figure 6A). The fluorescence intensity of 3,4-dihydroxyphenylalanine reduced by 74.9% when the proanthocyanidin concentration reached 50 μg/mL (curve 4 in Figure 6B). Concentrations of curves 0, 1, 2, 3, and 4 were 0, 20, 30, 40, and 50 μg/mL, respectively (Figure 6B). However, blue or red shifts were not found in the experiment. This indicated that the conformations of L-tyrosine and 3,4dihydroxyphenylalanine were unchanged. Curves labeled “a” were fluorescence emission spectra of proanthocyanidins with a concentration at 80 μg/mL. These results strongly proved that proanthocyanidins were the effective binding agents of L-tyrosine and 3,4-dihydroxyphenylalanine. Because of this, proanthocyanidins could efficiently stop the oxidation of the substrate. This could also make it clear that proanthocyanidins have a strong inhibitory effect on the tyrosinase catalytic reaction. In conclusion, this study demonstrated that avocado fruit proanthocyanidins were comprised of homo- and heteropolymers of procyanidins, prodelphinidins, propelargonidins, and procyanidin gallate with a degree of polymerization up to 36mers. They possessed structural heterogeneity in monomer units, substituent, and degree of polymerization. The results obtained from the enzyme assay revealed that these compounds were a potent, reversible, and competitive-type antityrosinase agent. Interactions of proanthocyanidins (particularly high polymer) with a substrate and dicopper catalytic center of tyrosinase were important mechanisms to explain their efficient inhibition. Structural similarity between proanthocyanidins and

Figure 6. (A) Relative fluorescence intensities of L-tyrosine in solution with different concentrations of proanthocyanidins. The concentrations of proanthocyanidins of curves 0, 1, 2, 3, and 4 were 0, 10, 30, 40, and 80 μg/mL. (B) Relative fluorescence intensities of 3,4dihydroxyphenylalanine in solution with different concentrations of proanthocyanidins. The concentrations of proanthocyanidins of curves 0, 1, 2, 3, and 4 were 0, 20, 30, 40, and 50 μg/mL, respectively. Curve a was the fluorescence intensity of proanthocyanidins when their concentration was 80 μg/mL.

fruit proanthocyanidins were competitive-type inhibitors (see Figure 2-III of the Supporting Information and Table 1). Plant extracts have attracted more and more attention because of their potent tyrosinase inhibition activity.23−25 In comparison to extracts obtained from Aloe vera L. gel,23 red koji,24 kiwifruit pericarp,4 and fruit stone of Chinese hawthorn,17 avocado fruit proanthocyanidins could better inhibit monophenolase and diphenolase activities. It could be speculated that strong inhibition of avocado fruit proanthocyanidins on the enzyme might mainly derive from structural similarity between the proanthocyanidin substrate of tyrosinase. In addition, a high polymer of avocado proanthocyanidins was considered as a important fator for the efficient inhibition. In our previous study, the proanthocyanidins with higher mDP showed better antityrosinase activity.4 In comparison to synthesized compounds, tyrosinase inhibitors obtained from natural sources have attracted more and more attention because of the food and cosmetic demands.26 Because of fact that proanthocyanidins are already in use for different medicinal properties, it is apparent that these compounds could well be applied in food, cosmetic, and pharmaceutical industries. Analysis of Fluorescence Quenching of Tyrosinase with Avocado Fruit Proanthocyanidins. Fluorescence quenching was used to evaluate the interaction of ligand (proanthocyanidins) and tyrosinase by using a Varian Cary Eclipse fluorescence spectrophotometer. Figure 4A showed the fluorescence emission spectra of tyrosinase with different concentrations of avocado fruit proanthocyanidins. The results showed that enzyme gave a strong emission peak at 337 nm and decreased inversely with an increasing concentration of proanthocyanidins (Figure 4B). Figure 4C, the Stern−Volmer plot, was selected to measure the Stern−Volmer quenching constants of the proanthocyanidins. The results showed a good linear Stern−Volmer plot with Kq and KSV values of (8 ± 0.2) × 105 (μg mL−1 s−1)−1 and (8 ± 0.2) × 10−3 (μg/mL)−1, respectively (Figure 4C and Table 2). Because of the fact that the KSV for dynamics cannot be larger than 100 L/M,15 the KSV that we obtained demonstrated that the type of fluorescence quenching mechanism between tyrosinase and avocado fruit proanthocyanidins was static. Figure 4D showed the plot of log[(F0 − F)/F] against log[Q]. From the intercept and the slope of this graph, values of KA and n were calculated to be (5 F


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the substrate of tyrosinase was the foundation of strong interactions. These findings played a significant role in both screening and designing great potential tyrosinase inhibitors. Our study revealed that proanthocyanidins purified from avocado fruit may have possible applications in agriculture, food, pharmaceutical, and cosmetic industries.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03099. Inhibition effect of avocado proanthocyanidins on monophenolase activity of mushroom tyosinase (Figure 1) and inhibitory effect (I), inhibitory mechanism (II), and inhibitory type (III) of avocado proanthocyanidins on the diphenolase reaction (Figure 2) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

The present investigation was supported by the Natural Science Foundation of China (21162012, 21362014, and 31260082), the Science and Technology Foundation of JianXi Province (20122BAB203007), and the Open Project Program of Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Jiangxi Normal University (KLFSKF-201419). Notes

The authors declare no competing financial interest.

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REFERENCES

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Avocado Proanthocyanidins as a Source of Tyrosinase Inhibitors

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