Condensed Tannins from Longan Bark as Inhibitor of Tyrosinase

College of Life Science and Key Laboratory of Small Functional Organic Molecule, Ministry of Education, Jiangxi Normal University, Nanchang, Jiangxi 3...
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Cite This: J. Agric. Food Chem. 2018, 66, 908−917

Condensed Tannins from Longan Bark as Inhibitor of Tyrosinase: Structure, Activity, and Mechanism Wei-Ming Chai,*,† Qian Huang,† Mei-Zhen Lin,† Chong Ou-Yang,† Wen-Yang Huang,† Ying-Xia Wang,† Kai-Li Xu,† and Hui-Ling Feng‡ †

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 ‡ Zigong Innovation Center of Zhejiang University, Zigong, Sichuan 643000, China ABSTRACT: In this study, the content, structure, antityrosinase activity, and mechanism of longan bark condensed tannins were evaluated. The findings obtained from mass spectrometry demonstrated that longan bark condensed tannins were mixtures of procyanidins, propelargonidins, prodelphinidins, and their acyl derivatives (galloyl and p-hydroxybenzoate). The enzyme analysis indicated that these mixtures were efficient, reversible, and mixed (competitive is dominant) inhibitor of tyrosinase. What’s more, the mixtures showed good inhibitions on proliferation, intracellular enzyme activity and melanogenesis of mouse melanoma cells (B16). From molecular docking, the results showed the interactions between inhibitors and tyrosinase were driven by hydrogen bond, electrostatic, and hydrophobic interactions. In addition, high levels of total phenolic and extractable condensed tannins suggested that longan bark might be a good source of tyrosinase inhibitor. This study would offer theoretical basis for the development of longan bark condensed tannins as novel food preservatives and medicines of skin diseases. KEYWORDS: condensed tannins, longan, structure, tyrosinase, inhibitor, mechanism

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units of propelargonidin) (Figure 1).12 These compounds can occur as highly complex mixtures of chemical structures because of monomer units, interflavonoid linkage (B-type and A-type), extent of polymerization, and modifications with nonflavonoid substituents.12−14,18 In consequence of the structure diversity, the analysis of condensed tannins is still difficult. In this study, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionization-full-mass spectrometry (ESI-FullMS), and high-pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were thus employed to illuminate the structure of longan bark condensed tannins. Longan (Dimocarpus longan Lour.) is a medicinal and food plant widely grown in China. Its fruits, seeds, and flowers are rich in phenolic compounds (e.g., proanthocyanidin, corilagin, gallic acid, and ellagic acid), which exhibit potent biological activity.19−21 However, there have been no reports of longan bark condensed tannins. In this study, the structure, antityrosinase activity, and mechanism of longan bark condensed tannins were therefore studied to provide a theoretical basis for their comprehensive utilization in the areas of food and medicine.

INTRODUCTION Tyrosinase (EC 1.14.18.1) catalyzes two crucial reactions in the melanogenesis, oxidation of monophenols to diphenols, and diphenols to o-quinone that ultimately transforms to melanin.1 Therefore, it is a key enzyme which is involved in deciding the skin and hair color.2,3 Its abnormal expression results in diverse skin diseases, including melanoma, freckles, and postinflammatory melanoderma.4 This enzyme is also an key enzyme that causes browning in fruits and vegetables, and the browning results in a loss of nutritive value.5 In addition, the enzyme plays a key part in the Parkinson’s disease.6 Therefore, tyrosinase inhibitors have received tremendous attention in the areas of food and medicine. A mass of inhibitors have been identified from synthetic sources, but they are not available because of their insolubility and high toxicity.7−9 Hence, it is considered that tyrosinase inhibitors from natural products should be expanded significantly for their water solubility, safety, and efficiency. It has reported that condensed tannins extracted from the fruit of avocado10 (mainly contain procyanidins) and leaves of Ficus altissima11 (main contain procyanidins) show better inhibition on the tyrosinase activity than arbutin. In this study, longan bark condensed tannins therefore are selected as a source of tyrosinase inhibitors. Condensed tannins are oligomeric and polymeric flavan-3ols.12 These compounds have attracted considerable research interest in recent years for their potential benefits to human health and for presenting diverse bioactivity, such as antioxidant, antitumor, and antienzymatic activities.13−15 Also, the biological abilities of condensed tannins largely depend on their structure.16,17 In general, the monomer of condensed tannins including catechin/epicatechin (monomer units of procyanidin), gallocatechin/epigallocatechin (monomer units of prodelphinidins), and afzelechin/epiafzelechin (monomer © 2018 American Chemical Society

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MATERIALS AND METHODS

Chemicals. Sephadex LH-20, cesium chloride, 2,5-dihydroxybenzoic acid, benzyl mercaptan, mushroom tyrosinase, L-tyrosine, and LDOPA (3,4-dihydroxyphenylalanine) were purchased from SigmaReceived: Revised: Accepted: Published: 908

November 27, 2017 December 30, 2017 January 9, 2018 January 9, 2018 DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structure of condensed tannins and flavan-3-ol monomer units. MALDI-TOF MS Analysis of Longan Bark Condensed Tannins. The MALDI-TOF MS method was executed according to our previous study.14 ESI-Full-MS Analysis of Condensed Tannins Purified from Longan Bark. ESI-Full-MS analysis was implemented by using Xevo G2 QT of MS (Waters, Milford, USA) in positive mode (ESI+). MS parameters were shown as follows: capillary voltage, 2000 V; sampling cone voltage, 100 V; source temperature, 120 °C; desolvation temperature, 400 °C; desolvation gas flow rate, 800 L/h; scan range, 500 to 2000 m/z. Injection volume was 200 μL, and detection flow rate was 20 μL/min. MS data and spectrum was obtained by MassLynx 4.0 (Waters). Thiolysis of Condensed Tannins with Benzyl Mercaptan and Reversed-Phase HPLC-ESI-MS Analysis of Product. Thiolysis of condensed tannins was accomplished according to previous report with minor modification.26 Briefly, 5 mg/mL of condensed tannins solution (50 μL, dissolved in methanol), 3.3% hydrochloric acid solution (50 μL, dissolved in methanol), and 5% benzyl mercaptan solution (100 μL, dissolved in methanol) were mixed. Reversed-phase HPLC-ESI-MS analysis of thiolysis product was executed in line with Zhou et al.22 Evaluation of the mean degree of polymerization (mDP) was performed in accordance with previous literature:16,27

Aldrich (St. Louis, MO). HPLC grade reagents including methanol, acetonitrile, and formic acid were acquired from Sinopharm (Sinopharm, Shanghai, China). Trypsin-EDTA, penicillin, streptomycin, and RPMI-1640 were acquired from Gibco (Germany). Diethyl pyrocarbonate (DEPC), 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Amresco. All analytical grade reagents for extraction and purification including acetone, ethyl acetate, petroleum ether, and methanol were acquired from Sinopharm. Content Assay of Total Phenolics and Extractable Condensed Tannins. The Folin-Ciocalteu method22 was selected for measuring the total phenols content of longan bark. In a word, sample solution (0.2 mL), distilled water (0.3 mL), Folin-Ciocalteu reagent (0.5 mL, 1 N), and Na2CO3 solution (2.5 mL, 20%) were mixed and shaken well. The results were read at 725 nm. Catechin was used as total phenolics calibration. The content of extractable condensed tannins was decided by the butanol−HCl method.23 In brief, sample solution (1 mL) and butanol−HCl (v:v, 95:5, 6 mL) were mixed. The absorbances were recorded at 550 nm. All of the experiments were repeated three times. The purified condensed tannins fraction of longan bark was used for condensed tannins calibration [condensed tannins content = 100%, assessed by liquid chromatography−mass spectrometry (LC−MS) after thiolysis].24 Sample Preparation, Extraction, and Purification of Condensed Tannins. Longan barks were gathered at Xiamen University (Xiamen, Fujian Province, China) in June 2015. They were immediately cleaned (the longan barks were washed with tap water and then with distilled water three times) in the laboratory. Then we used a vacuum freeze-drying machine to obtain freeze-dried powders of the longan barks. The extraction method used was as described in our previous report with some modifications.10 In brief, powders of longan barks were ultrasonically extracted with 70% acetone−water in a beaker. The beaker was sealed with plastic wrap during the extraction process. To prevent the autoxidation/oxygen contact of condensed tannins, 0.05% ascorbic acid was added into 70% acetone−aqueous simultaneously. Petroleum ether and ethyl acetate were used as extractant. Then Sephadex LH-20 was used to get purified condensed tannins of longan barks. Purified process was carried out according toprevious reports.10,25

mDP = (total area of the extender units) /(total area of the terminal units) + 1 Inhibitory Effect of Longan Bark Condensed Tannins on the Monophenolase Activity. For the monophenolase activity assay, Ltyrosine was used as a substrate. The reaction system contained 0.5 mL of L-tyrosine (12 mM), 0.75 mL of Na2HPO4−NaH2PO4 buffer (50 mM), 0.1 mL of inhibitor (dissolved in water) with different concentrations, 1.55 mL of H2O, and 0.1 mL of mushroom tyrosinase solution (dissolved in water). They were well mixed and read at 475 nm. All experiments were repeated three times. Inhibition of Longan Bark Condensed Tannins against the Diphenolase Activity. For the diphenolase activity assay, L-DOPA was selected as the substrate. In total, 0.3 mL of L-DOPA (0.5 mM), 0.75 mL of Na2HPO4−NaH2PO4 buffer, 1.8 mL of H2O, and 0.1 mL of inhibitor were mixed. Finally, 0.05 mL of enzyme solution (0.2 mg/ mL) was added. In the experiment, the time-interval kinetic 909

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

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Journal of Agricultural and Food Chemistry measurement for the inactivation time-course was used to check activity change. The initial slope (ΔOD/ΔT, ΔT = 30 s) of the kinetic curve was used as an indicator of diphenolase activity. Absorption was detected at 475 nm on Beckman DU-800 (California). H2O was used as a control. Inhibitory Mechanism of Longan Bark Condensed Tannins on the Diphenolase Activity. In the reaction system, the concentration of the enzyme was changed to study the inhibitory mechanism. The plots of the remaining enzyme activities versus the enzyme concentrations in the presence of inhibitor with different concentrations gave a group of straight lines. The inhibition was considered to be reversible when they meet at the origin. Also, the inhibition was irreversible when they are parallel lines. Inhibition Type and Inhibition Constants of Longan Bark Condensed Tannins on the Diphenolase Activity. The assessment of inhibition type and inhibition constant was performed in line with our previous study.28 In a word, the Lineweaver−Burk plot was used to decide inhibition type. The second plots of the apparent Km/ Vm or 1/Vm versus the inhibitor concentration were used to determine inhibition constants. Cell Culture and MTT Assay. RPMI 1640 medium supplemented with 10% of heat-inactivated fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin was used to culture B16 mouse melanoma cells. The cells were cultured at 37 °C in a humidified atmosphere under 5% CO2.29 For measuring the activity of B16 mouse melanoma cells, MTT assay was carried out.30 Inhibition Effect of Longan Bark Condensed Tannins on the Intracellular Tyrosinase Activity of B16 Mouse Melanoma Cells. The inhibition effect of longan bark condensed tannins on the intracellular tyrosinase activity of B16 mouse melanoma cells was detected in line withprevious reports.29,31 In short, B16 cells were cocultured with longan bark condensed tannins for 48 h. After that, the cells were lysed with lysis buffer. The supernatants were collected after cellular lysates were centrifuged. Protein content was determined using the bicinchoninic acid (BCA) protein assay kit. The supernatant (0.1 mL) of cell extract, L-DOPA (0.9 mL, 0.5 mM), and phosphate buffer (pH 6.8) were mixed well to measure tyrosinase activity. UV absorptions were read at 475 nm on Beckman DU-800. Inhibition of Longan Bark Condensed Tannins on the Intracellular Melanin Content of B16 Mouse Melanoma Cells. The inhibition method used was as described in Chen et al. with some modifications.31 B16 cells were cocultured with longan bark condensed tannins for 48 h. After that, the cells were collected. An aliquot was used for protein determination, and the remaining cells were centrifugated at 10 000 g for 15 min. The protein contents were determined using the BCA protein assay kit, and then the remaining cells were lysed by using 0.5 mL of NaOH (5 M) at 100 °C for 1 h. Next, a part of crude cell extracts (0.2 mL) were moved into a 96-well plate. Melanin content were determined according to the OD value at 405 nm. Molecular Docking of Tyrosinase with the Ligands. Protein− ligand docking was implemented by using ActoDock tools. The X-ray structure of oxy tyrosinase from Agaricus bisporus (2Y9W)32 was selected as the initial model for docking simulation. ChemBioDraw Utra 12.0 was used to prepare the three-dimensional (3D) structures of the ligands. Water molecules were first removed. Then Gasteiger charges and polar hydrogen atoms were added to the enzyme molecule before docking. The parameters for docking and calculation were default. The docked mode were acquired according to the conformation which needs the lowest energy.33 Statistical Analysis. SPSS 19.0 was used for the calculations. Experimental results were expressed as mean ± standard deviation of three replications.

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Table 1. Yield, Content of Total Phenolics, and Extractable Condensed Tannins sample

yield (%)

total phenolics (g/kg dry weight)

extractable condensed tannins (g/kg dry weight)

condensed tannins

5.32

250.6 ± 9.8

198.3 ± 8.7

Figure 2. (A) MALDI-TOF positive-ion (Cs+) reflectron mode mass spectra of the condensed tannins from longan bark. (B) Magnified MALDI-TOF mass spectra of 3-mers, 4-mers, and 5-mers.

acetate) and Sephadex LH-20 column chromatography. Finally, we obtained 532 mg of purified condensed tannins. Therefore, the total extraction yield was 5.32% (referred to as dry bark weight) (Table 1). Compared with the yield obtained from pericarp of Clausena lansium,29 fruit stones, and pericarps of Litchi chinensis,22 the longan bark showed an approximate result. Polyphenol compounds (including condensed tannins) were widely studied in recent years due to their diverse biological activity (e.g., antioxidant, anticancer, and enzyme inhibition).13,14,34 A high level of total phenolics (250.6 ± 9.8 mg/g) (Table 1) and extractable condensed tannins (198.3 ± 8.7 mg/g) (Table 1) in longan bark revealed that this material could be selected as the source of condensed tannins. Structure Analysis of Longan Bark Condensed Tannins. Mass spectrometry is a good technique to track the molecular distribution. To this end, two different mass spectrometric techniques were used to evaluate the molecular structure of longan bark condensed tannins. ESI-Full-MS provided structural details of oligomer of condensed tannins (the detection range was from 50 to 2000 Da); the MALDITOF MS technique offered qualitative and quantitative information on higher molecular weight condensed tannins. Additionally, HPLC-ESI-MS analysis coupled with thiolysis gave the structural information on monomer units. Therefore, the united use of different mass techniques would obtain a more comprehensive and accurate structure outline of longan bark condensed tannins. MALDI-TOF MS Analysis. MALDI-TOF MS technique was an appropriate method for identification of condensed

RESULTS AND DISCUSSION

Extraction Yield, Contents of Total Phenolics, and Extractable Condensed Tannins. Dry powder (10 g) of longan bark was ultrasonically extracted with 70% acetone, purified by solvent extraction (petroleum ether and ethyl 910

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

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Figure 3. ESI-Full-MS positive mode mass spectrum of longan bark condensed tannins.

Figure 4. (A) Reversed-phase HPLC-ESI-MS chromatograms of condensed tannins from longan bark after thiolytic degradation. G, gallocatechin; EG, epigallocatechin; C, catechin; EC, epicatechin; AF, afzelechin; EAF, epiafzelechin. 911

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

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Journal of Agricultural and Food Chemistry

Figure 5. Inhibition effects of condensed tannins from longan bark on enzyme activity of mushroom tyosinase: (A) progress curves for the oxidation of L-tyrosine by the enzyme and (B) effects on the steadystate rate for the oxidation of tyrosine. The concentrations of condensed tannins for curves 0−5 were 0, 15, 30, 45, 60, and 90 μg/ mL, respectively. The IC50 was expressed as the mean of triplicate determinations ± standard deviation.

tannins which showed structural heterogeneity.13,35 Figure 2A showed the MALDI-TOF mass spectra of longan bark condensed tannins (the molecular weight ranging from 900 to 5500 Da). The MALDI-TOF mass spectra had peaks which increased equably at a mass distance of 288 Da (996.73, 1284.95, 1573.06, 1861.03, 2148.86, 2436.55, 2725.11, 3013.12, 3301.19, 3589.21, and 3877.16 Da). The increment of 288 Da was in accordance with the molecular weight of one (epi)catechin (EC/C), which reflected that the main structural unit of longan bark condensed tannins was EC/C. Therefore, it could be further concluded that condensed tannins from longan bark were mainly procyanidins. For each polymers, substructures with higher or lower 16 Da were also detected (Figure 2A). These masses were heteropolymers of flavan-3-ol units, which demonstrated the presence of (epi)gallocatechin (EGC/GC) and (epi)afzelechin (EAF/AF). The above findings indicated the existence of prodelphinidins and propelargonidins in longan bark condensed tannins. Compared with procyanidins, there are obviously fewer prodelphinidins and propelargonidins. Besides the series of peaks described above, 152 Da mass distances following the major set of peaks were also observed (Figure 2A), which are in keeping with the addition of one galloyl group at the heterocyclic C-ring. In addition, 132 Da mass distance following the main set of peaks was produced by synchronous attachment of two Cs+ and absence of a proton [M + 2Cs + 2H]+.36 Specifically, a series of peaks which followed the main set of peaks with 120 Da mass distance were also discovered in the mass spectrum (Figure 2A). This result suggested the presence of acyl derivatives (probably corresponding to p-hydroxybenzoate derivative).37 Further studies were needed to better understand this finding. Furthermore, the A-type interflavan linkages of 2 Da less than the B-type were also detected in the mass spectrum (Figure 2B). In accordance with the molecular weight of the smallest and largest tannin oligomer (996.73 and 3877.16 Da), it could be concluded that the distribution of degree of polymerization of longan bark condensed tannins was 3-mers to 13-mers. In summary, these results demonstrated that the condensed tannins from longan bark were a complicated mixture of procyanidins, prodelphinidins, propelargonidins, and their derivatives. These polymers have structural heterogeneity which derived from the monomeric unit, interflavan linkage, molecular weight (DP), and substituent. What needs to be pointed out was that longan bark condensed tannins mainly included procyanidin and was rich in A-type linkage. The structures of longan bark condensed tannins were therefore

Figure 6. Inhibitory effect (A), mechanism (B), type and constant (C) of longan bark condensed tannins on the diphenolase activity of tyrosinase. The concentrations of condensed tannins for curves 0−4 were 0, 20, 40, 60, and 80 μg/mL, respectively. The inhibition type assay was studied by the Lineweaver−Burk plot, and the inhibition constant was determined by the second plots of the apparent Km/Vm or 1/Vm versus the concentration of the inhibitor. The IC50 was expressed as the mean of triplicate determinations ± standard deviation.

successfully characterized by using the MALDI-TOF MS method for the first time. ESI-Full-MS Analysis. Longan bark condensed tannins were also characterized with the ESI-Full-MS system in positive mode. Leucine enkephalin was used as an internal standard. As shown in Figure 3, the first sequence of peaks were [M + H]+ ion peaks of B-type dimeric (m/z 579 Da), A-type trimeric (m/ z 865 Da), A-type tetrameric (m/z 1153 Da), and A-type pentameric (m/z 1441 Da) procyanidins, respectively.38 Simultaneously, a second sequence of peaks arranged from m/z 887 to 1751 Da were also observed (Figure 3A). These peaks increased equably at a mass distance of 288 Da which was in line with the addition of one EC/C monomer unit. In fact, these signals were [M + Na]+ ion peaks of A-type trimeric (m/z 887 Da), A-type tetrameric (m/z 1175 Da), A-type pentameric 912

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

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AF benzylthioether (395, [M − H] − ), A-type dimer benzylthioether (697 Da, [M − H]−), and A-type trimer benzylthioether (985 Da, [M − H]−).16,22 What needs to be pointed out was that the peak for A-type dimer (∼11 min) was extremely weak, but this did not reflect the actual content of it. The probable reason was that most of A-type dimer had been extracted by using ethyl acetate. The above findings demonstrated again the coexistence of procyanidins (A-type and B-type), propelargonidins, and prodelphinidins in the longan bark condensed tannins. However, there were no EC/C gallate in the chromatogram, and we speculated that they were few (with a weak intensity) in the longan bark. These results recommended that the diversity of longan bark condensed tannins derived from their monomer units and interflavan linkage, which offered a good supplement to MALDI-TOF MS and ESI-Full-MS analyses. In addition, the mDP of longan bark condensed tannins was evaluated to be 3.5 ± 0.2. Compared with the mDP obtained from the fruit of avocado (12.3),10 fruit stones (15.4) and pericarp (5.8) of litchi,22 pericarp of wampee (13),29 longan bark condensed tannins showed a smaller average molecular weight. Effect of Longan Bark Condensed Tannins on the Monophenolase. Monophenolase is the limited enzyme of melanogenesis.39 Therefore, the inhibitor of monophenolase could be a potential medicine for treating skin disease. In this study, the inhibitory effect of longan bark condensed tannins on monophenolase was thus studied using L-tyrosine as the substrate (Figure 5A). The steady-state rate reduced distinctly with the increase of condensed tannins concentration (Figure 5B). The condensed tannins concentration resulted in 50% loss of the steady-state rate (IC50) and was estimated to be 43.7 ± 0.3 μg/mL. Therefore, it could be concluded that longan bark condensed tannins were good inhibitors of monophenolase. The efficient inhibition may largely depended on the structural feature of condensed tannins. These polyphenol compounds all possessed a benzene ring and many hydroxyl groups. This structural feature was very similar to the construction of tyrosinase substrate (L-tyrosine). Therefore, the presence of condensed tannins might intensely hinder the oxidation process of substrate which is catalyzed by tyrosinase. Compared with condensed tannins from the fruit of avocado10 (IC50 = 40 ± 1.2 μg/mL), pericarp of Clausena lansium28 (IC50 = 23.6 ± 0.3 μg/ mL), and fruit stone of hawthorn40 (IC50 = 37 ± 0.5 μg/mL), the inhibition of longan bark condensed tannins on the monophenolase was slightly weaker. This might arise from abundant A-type linkage and oligomer in the longan bark condensed tannins. In our previous research, condensed tannins from avocado and Clausena lansium were composed of B-type polymer with greater mDP. In summary, longan bark condensed tannins showed strong inhibition on the monophenolase activity and might be a good candidate for developing a new medicine and food preservative. Inhibitory Effect, Mechanism, and Type of Longan Bark Condensed Tannins on Diphenolase. Diphenolase was also a key enzyme for melanin biosynthesis. In the current study, the impact of the longan bark condensed tannins on diphenolase activity was thus detected using L-DOPA as substrate. As shown in Figure 6A, with an increased concentration of longan bark condensed tannins, the relative activity of diphenolase decreased quickly. The condensed tannins concentration resulted in 50% loss of enzyme activity and was calculated to be 11.5 ± 0.8 μg/mL (IC50). In consideration of the mean molecular weight (288 * mDP) of

Figure 7. Effects of condensed tannins from longan bark on cell activity, tyrosinase activity, and cellular melanin content of B16 cells. Cells were treated with compounds for 48 h at various concentrations. All the experiments were repeated three times.

(m/z 1463 Da), and A-type hexameric (m/z 1751 Da) procyanidins, respectively. It could be further concluded that the main constituent of longan bark condensed tannins was procyanidins. When the 3-mers were enlarged (insert of Figure 3A), substructure signals with higher or lower 16 Da were detected (m/z 871 and 903 Da), they were produced by the loss or the addition of an hydroxyl group at the 5′ position of the B-ring. Similar phenomenon was found when 4-mers was magnified (m/z 1159 and 1191 Da).38 These results revealed the presence of EGC/GC and EAF/AF. It therefore could be further concluded that longan bark condensed tannins contain propelargonidins and prodelphinidins. In addition to the signals indicated above, 152 Da mass distances following the first series peaks (m/z 1019 and 1307 Da) and the second series peaks (m/z 1039 and 1327 Da) were also observed (insert of Figure 3A). These finding demonstrated the addition of one galloyl group to the heterocyclic C-ring of condensed tannins from longan bark.38 Furthermore, A-type condensed tannins were also found when polymers (3-mers, 4-mers, and 5-mers) were enlarged (Figure 3B). In addition, 2-mers to 6-mers were detected in the mass spectra. In summary, longan bark condensed tannins were composed of procyanidins, propelargonidins, prodelphinidins, and procyanidin gallates. On the basis of ESI-Full-MS result, this technique was very sensitive for the detection of A-type condensed tannins. These results agreed well with the MALDI-TOF MS analysis. The above results demonstrated that ESI-MS method was a effective tool in revealing condensed tannins structure (specifically for A-type linkage) and determining the presence of oligomeric polymer. However, compared with MALDI-TOF MS analysis, condensed tannins with higher molecular weight were difficult to detect with good precision.38 Reversed-Phase HPLC-ESI-MS Analysis after Thiolysis. Thiolysis combined with the reverse phase HPLC-ESI-MS (negative mode) analysis was a powerful tool in analyzing the structure of condensed tannins purified from plants.14,16,26 The chromatogram of longan bark condensed tannins was shown in Figure 4. According to standards and molecular weight obtained from ESI-MS, the terminal units were identified as GC (305 Da, [M − H]−), EGC (305 Da, [M − H]−), C (289 Da, [M − H]−), EC (289 Da, [M − H]−), AF/EAF (273 Da, [M − H]−), and A-type dimer (575 Da, [M − H]−). Extended units were confirmed to be EGC/GC benzylthioether (427 Da, [M − H]−), EC/C benzylthioether (411 Da, [M − H]−), EAF/ 913

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

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Figure 8. Molecular docking result of the ligands with tyrosinase residues.

The inhibitory mechanism of longan bark condensed tannins on the diphenolase was further studied by changing the enzyme concentration. As shown in Figure 6B, a group of straight lines intersected at the origin, indicating that the inhibition was reversible. Therefore, the longan bark condensed tannins did

longan bark condensed tannins, the IC50 values could be transformed into about 0.01−0.013 μM. Quercetin was generally considered a good tyrosinase inhibitor with a IC50 value of 0.13 mM.41,42 Therefore, longan bark was a better source of diphenolase inhibitor. 914

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

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Journal of Agricultural and Food Chemistry

concentration of condensed tannins reached 200 μg/mL. These results showed that there was a critical value (the critical concentration of condensed tannins without influence on the cell proliferation) during the effection process of longan bark condensed tannins on the cell proliferation. The cells were activated when the condensed tannins concentration below the critical value and were inhibited when the condensed tannins concentration above the critical value. The effect of longan bark condensed tannins on the cellular tyrosinase activity and melanin content of B16 cells were further studied. The result revealed that intracellular tyrosinase activity was obviously reduced with the increasing concentrations of longan bark condensed tannins. When the concentration of condensed tannins reached 200 μg/mL, the intracellular tyrosinase activity and melanin content of B16 cells decreased to 40.3 ± 1.5% and 45.2 ± 1.3%, respectively. The above results indicated that the condensed tannins from longan bark could inhibit the cell proliferation, intracellular tyrosinase activity, and melanin production of B16 mouse melanoma cells. Molecular Docking. Molecular docking, a complementary application, has recently been widely employed to explore the probable interaction between inhibitor and enzyme.33,44 To better understand the interaction mechanism of the longan bark condensed tannins with tyrosinase, molecular docking was implemented using ActoDock tools. In this study, (epi)afzelechin (EAF/AF), (epi)catechin (EC/C), (epi)gallocatechin (EGC/GC), B-type dimer, A-type dimer, and A-type trimer (3 epicatechin) were selected as ligands. As shown in Figure 8 and Table 2, the noncovalent interactions between the ligands and tyrosinase were proved mainly to be hydrogen bonds and hydrophobic interaction. The hydrogen bonds were generated by AF with the B chain of amino residues Gln44, Glu173, and His178; EAF with B chain of amino residues Glu67, Gln74, Thr324, and Gly326; C with B chain of amino residues Glu356; EC with A chain of amino residues Gln44 and Lys158; GC with B chain of amino residues Thr308, Asp312, and Glu356; EGC with A chain of amino residues Asp312, Glu 356, and Lys379; B-type dimer with B chain of amino residues Ala219, Gly223, and Pro270; A-type dimer with B chain of amino residues Tyr343; A-type trimer with A chain of amino residues Ala45; respectively. Moreover, the hydrophobic interactions were produced by C with the B chain of amino residues Trp358, Lys376, and Lys379; EC with the A chain of amino residues Lys180; B-type dimer with the B chain of amino residues Ala220, Phe224, Trp227, and Leu265; respectively. In addition, it was also found that electrostatic interactions were formed between A-type trimer and the A chain of amino residue Glu173. These results showed that the hydrogen bond and hydrophobic and electrostatic interactions were intrinsic driving forces of the inhibition. Among these three intermolecular interactions, the hydrogen bond played the most important role in the inhibition process. This offered an intrinsic mechanism to understand the efficient inhibition of longan bark condensed tannins on the tyrosinase. In conclusion, this study illustrated that longan bark condensed tannins contain prodelphinidins, propelargonidins, procyanidins, and their acyl derivatives (galloyl and phydroxybenzoate). They had structural diversity which derived from monomeric unit, interflavan linkage, DP, and substituent. These condensed tannins were efficient, reversible, and mixed tyrosinase inhibitors. Interactions between tyrosinase and inhibitor driven by hydrogen bond and hydrophobic and electrostatic interactions offering a feasible molecular mecha-

Table 2. Molecular Docking Results of the Ligands with Tyrosinase Residuesa ligand AF EAF C EC GC

hydrogen bond Gln44, Glu173, His178 (B) Glu67, Gln74, Thr324, Gly326 (B) Glu356 (B)

Gln44, Lys158 (A) Thr308, Asp312, Glu356 (B) EGC Asp312, Glu 356, Lys379 (A) B-type dimer Ala219, Gly223, (2EC) Pro270 (B) A-type dimer Tyr343 (B) (2EC) A-type Ala45 (A) trimer (3EC)

hydrophobic interaction

electrostatic interaction

Trp358, Lys376, Lys379 (B) Lys180 (A)

Ala220, Phe224, Trp227, Leu265 (B)

Glu173 (A)

a

AF, afzelechin; EAF, epiafzelechin; E, catechin; EC, epicatechin; GC, gallocatechin; EGC, epigallocatechin; A,B, A or B chain of tyrosinase.

not reduce the amount of diphenolase, but inhibited its enzyme activity.41 The inhibition type on the diphenolase by longan bark condensed tannins was determined using a Lineweaver−Burk plot. As shown in Figure 6C-I, the plot of 1/v versus 1/[ LDOPA] gave a group of straight lines which intersected in the second quadrant. Thus, longan bark condensed tannins were competitive−uncompetitive mixed competitive inhibitors. The inhibition constants of condensed tannins binding with the free enzyme (KI) and with the enzyme−substrate complex (KIS) were determined to be 9.4 ± 0.1 and 18.6 ± 0.2 μg/mL, respectively (Figure 6C-II,III). The value of KI was lower than that of KIS, indicating the affinity of the condensed tannins with free enzyme was stronger than that of the enzyme−substrate complex.43 Compared with the inhibition type (competitive) of avocado condensed tannins (mainly contain B-type procyanidin),10 they were different. We speculated that condensed tannins tend to produce a competitive inhibition when they include only one type of tannin (e.g., B-type procyanidin). It was extremely likely that condensed tannins with the same type tend to behave the same way in the inhibition. Because they had very similar structures. However, when plant tannins emerge with more diversity (e.g., the coexistence of A-type and B-type condensed tannins; the coexistence propelargonidin, prodelphinidins, and procyanidin) in the structure, they tend to form a mixed competitive inhibition. This might arise from mutual interference of condensed tannins with different structures. It should be pointed out that the relation between structure of condensed tannins and inhibition type still needs more evidence. Cell Tests. The cytological effect of longan bark condensed tannins on the proliferation of B16 mouse melanoma cells was carried out by using the MTT method. As shown in Figure 7, the condensed tannins promoted the proliferation of the melanoma cell when their concentration was set at 50 μg/mL. However, these compounds start inhibiting the multiplication of the cells when the concentration reached 100 μg/mL and showed a concentration-dependent relationship after that. The remaining relative activity decreased to 32.7 ± 7.8% when the 915

DOI: 10.1021/acs.jafc.7b05481 J. Agric. Food Chem. 2018, 66, 908−917

Article

Journal of Agricultural and Food Chemistry

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nism to explain the inhibition of longan bark condensed tannins on tyrosinase. Longan bark condensed tannins also showed good inhibitions on cell proliferation, cellular tyrosinase activity, and melanogenesis of B16 mouse melanoma cells. This study demonstrated that longan bark condensed tannins were powerful tyrosinase inhibitors and offered a theoretical basis for their comprehensive utilization in the areas of food and medicine.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The present investigation was supported by the Natural Science Foundation of China (Grant No. 31501414), Natural Science Fund of Jiangxi Province (Grant No. 20171BAB214019), Education Department Project of Jiangxi Province (Grant No. GJJ150302), and Provincial Graduate Innovation Fund Project of Jiangxi Province (Grant No. YC2017-S151). Notes

The authors declare no competing financial interest.

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