Structural Analysis of Proanthocyanidins Isolated from Fruit Stone of

Article pubs.acs.org/JAFC

Structural Analysis of Proanthocyanidins Isolated from Fruit Stone of Chinese Hawthorn with Potent Antityrosinase and Antioxidant Activity Wei-Ming Chai,† Chih-Min Chen,† Yu-Sen Gao,† Hui-Ling Feng,† Yu-Mei Ding,† Yan Shi,† Han-Tao Zhou,‡ and Qing-Xi Chen*,† †

State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361005, China Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen 361005, China

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ABSTRACT: Proanthocyanidins were isolated from fruit stone of Chinese hawthorn (Crataegus pinnatif ida Bge. var. major N.E.Br.). Their structures were analyzed and elucidated by methods of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS). The results demonstrated that these compounds are complicated mixtures of homo- and heteropolymers consisting of procyanidin/procyanidin gallate and prodelphinidin. They possessed structural heterogeneity in monomer units, polymer length, and interflavan linkage (A-type and B-type). Their antityrosinase and antioxidant activity were then investigated. The results revealed that they can inhibit tyrosinase activities, including the monophenolase activity and the diphenolase activity. In addition, proanthocyanidins possessed potent antioxidant activity. Our studies revealed that proanthocyanidins isolated from fruit stone of Chinese hawthorn may be applied in food, agriculture, pharmaceutical, and cosmetic industries. KEYWORDS: hawthorn proanthocyanidins, structural analysis, MALDI-TOF MS, HPLC-ESI-MS, antityrosinase activity, antioxidant activity

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INTRODUCTION Proanthocyanidins are polyphenol compounds. They are most commonly found in the plants and have aroused considerable interest and received more and more attention in recent years due to their bioactive functions. These compounds are composed of flavan-3-ol monomer units (Figure 1), which are joined together through C4−C8 or C4−C6 bonds to Btype oligomers and high polymers.1 When C2 and C7 are linked by an additional ether linkage, these compounds are Atype proanthocyanidins (Figure 1).2 The structural variability of monomer units, interflavan linkage, and distribution of polymerization degree contribute to the diversity of proanthocyanidins.2,3 The qualitative and quantitative analysis of these compounds remains very challenging because of their structural diversity and complexity. In our previous report, proanthocyanidins isolated from stem bark, fruit, and leaf of Delonix regia were found with potent tyrosinase inhibitory activity.4 Tyrosinase (EC 1.14.18.1) is a kind of oxidase that contains copper and possesses two successive catalytic activities: the monophenolase activity (hydroxylation of monophenols to o-diphenols) and diphenolase activity (the oxidation of o-diphenols to o-quinones).5 It causes melanization in animals and browning in plants.6,7 In addition, tyrosinase is important in normal developmental processes in insects.8 This indicates that tyrosinase inhibitors are potential insect control agents as well as whitening agents, food additives, and medicine. Plants are rich in bioactive compounds that have few side effects; thus researchers have shown increasing interest in employing them as a source of tyrosinase inhibitors.9 © 2013 American Chemical Society

Hawthorn is one of the most popular medicinal and food materials in the world. Its fruits, leaves, bark, and flowers contain significant amounts of phenolic compounds, which exhibit potent antioxidant activity.10−12 Most of these compounds belonged to proanthocyanidins.13,14 In addition, Svedström et al. found that oligomeric proanthocyanidins in different tissue of hawthorn range from 2-mers to 6-mers.15 However, detailed structure information and antityrosinase and antioxidant activity of purified proanthocyanidins extracted from Chinese hawthorn fruit stone have not been reported. Our study primarily aims to provide thorough structural analysis of proanthocyanidins from fruit stone of Chinese hawthorn using reflectron pattern MALDI-TOF MS, linear pattern MALDI-TOF MS, reversed-phase HPLC-ESI/MS, and normal-phase HPLC-ESI/MS analysis. These techniques are initially applied on Chinese hawthorn fruit stone proanthocyanidins to clarify the elementary units, distribution of DP, and type of linkage. The antityrosinase and antioxidant activity information on these compounds are also provided in this study.

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MATERIALS AND METHODS

Chemicals. HPLC standards (catechin and epicatechin), Sephadex LH-20, trifluoroacetic acid, benzyl mercaptan, Amberlite IRP-64 cation-exchange resin, CsCl, and 2,5-dihydroxybenzoic acid were Received: Revised: Accepted: Published: 123

July 23, 2013 December 7, 2013 December 8, 2013 December 8, 2013 dx.doi.org/10.1021/jf405385j | J. Agric. Food Chem. 2014, 62, 123−129

Journal of Agricultural and Food Chemistry

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as the matrix, and Cs+ was selected as the cationization reagent. Amberlite IRP-64 cation-exchange resin was dissolved in deionized water, which was used to remove ions in the sample and matrix solution. The mixture volumetric ratio of the sample solutions and the matrix solution was 1:3. The final mixed solution (1 μL) was then dripped to the steel target.16 Reversed-Phase HPLC-ESI-MS Analysis Followed Thiolysis Reaction. With an Agilent 1200 system (Agilent, Palo Alto, CA, USA) coupled to a QTRAP 3200 (Applied Biosystems, Foster, USA), reversed-phase HPLC-ESI-MS analysis was conducted. The column employed in the analysis was a 250 mm × 4.6 mm i.d., 5 μm, LiChrospher 100 RP-18, with a 4 mm × 4 mm i.d. guard column of the same material (Elite, Dalian, China). The linear gradient elution process was performed as follows (two solvents were used as mobile phase, 0.5% trifluoroacetic acid and acetonitrile): 0−45 min, 12−80% acetonitrile; 45−50 min, 80−12% acetonitrile. The flow rate was kept at 1 mL/min, and the chromatogram was detected at 280 nm. In addition, instrument parameters for MS analysis in the negative ion pattern were set as follows: ion spray voltage, 4.5 kV; declustering potential, 50 V; entrance potential, 10 V; curtain gas, 20 psi; source temperature, 400 °C. Normal-Phase HPLC-ESI-MS Analysis. Analysis was performed on a 250 mm × 4.6 mm i.d., 5 μm Silica Luna (Phenomenex, Darmstadt, Germany). The column temperature was controlled at 35 °C. The HPLC and MS system were the same as reversed-phase HPLC-ESI-MS analysis. Two solvents were used: A = water/acetic acid/methanol/dichloromethane (1:1:7:41, v/v/v/v) and B = water/ acetic acid/methanol/dichloromethane (1:1:5:43, v/v/v/v). The linear gradient elution program was as follows: 0−20 min, 0−13.5% B; 20− 50 min, 13.5−29.2% B; 50−55 min, 29.2−100% B; 55−60 min, 100% B.17 Conditions for MS analysis were the same as the reversed-phase HPLC-ESI-MS analysis described previously. Assay of the Enzyme Activity. Enzyme reaction was performed in compliance with the program of our previous study.18 L-Tyrosine (2.17 mg/mL) was used as substrate for monophenolase activity assay and 3,4-dihydroxyphenylalanine (1 mg/mL) was used as substrate for diphenolase activity assay. The substrate and tyrosinase were mixed well in the absence or presence of the sample (3 mL reaction system) with different concentrations. The final concentrations of tyrosinase were 33.33 μg/mL for monophenolase activity assay and 6.67 μg/mL for diphenolase activity assay. Their enzyme activities were recorded on a Beckman UD-800 spectrophotometer (Beckman coulter, Pasadena, California, USA) at 475 nm. Lag time (the time for the reaction system reaching steady-state rate) and steady-state rate (a constant rate) were employed to measure the effect of proanthocyanidins on the enzyme activity of monophenolase. The effect of proanthocyanidins on the diphenolase reaction was expressed as percentage. Fluorescence Analysis. Varian Cary Eclipse fluorescence spectrophotometer was used to record the fluorescence spectra. The reaction system (3 mL) contained 300 μL of mushroom tyrosinase solution, 300 μL of sample solution with different concentrations, and 2.4 mL of sodium phosphate buffer (50 mM, pH 6.8). Excitation wavelength was 290 nm. All the tests were peformed at room temperature. Determination of Cu2+ Chelating Activity. The reaction system (3 mL) contained 100 μL of sample (2 mg/mL) solution, 100 μL of copper sulfate solution with different concentrations (25, 50, 100 μM), and 2.8 mL of sodium phosphate buffer (50 mM, pH 6.8). The mixture was shaken well and kept at 25 °C for 10 min prior to UV−vis analysis. DPPH Radical Scavenging Activity Assay. DPPH analysis was performed in accordance with the report of Brand-Williams et al.19 The reaction system was comprised of 0.1 mL of sample solution (dissolved in methanol) and 3 mL of DPPH solution (25 mg/L in methanol). The results were detected at 517 nm after the reaction mixture was shaken well and kept at 25 °C for 30 min. The scavenging effect was determined on the basis of the following equation:

Figure 1. Chemical structure of flavan-3-ol monomer units and proanthocyanidins. obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Mushroom tyrosinase, L-tyrosine, 3,4-dihydroxyphenylalanine, dimethyl sulfoxide, DPPH, ABTS, TPTZ, AAPH, and ascorbic acid, were also obtained from Sigma-Aldrich. The solvents with standard analytical purity were acquired from Sinopharm (Shanghai, China). Acros Organics (New Jersey, USA) is the company that provided the fluorescein and trolox. Plant Material and Sample Preparation. Hawthorn fruits at commercial maturation were picked on the campus of Jiangxi Normal University (Nanchang, Jiangxi Province, China) in July. Fruit stones were isolated manually and immediately freeze-dried and then were ground to fine powders. The powders were placed at −80 °C before next analysis. Extraction and Purification of Hawthorn Fruit Stone Proanthocyanidins. The powders (10.0 g) were ultrasonically extracted three times (each 200 mL) with acetone−water (7:3, v/v) solution containing 0.1% ascorbic acid at 25 °C. The acetone was eliminated in a rotary evaporator at 38 °C. Chlorophyll and lipophilic compounds remained in aqueous fraction were eliminated by extracting them with petroleum ether three times (each 150 mL). The remaining aqueous fraction was purified by using chromatography on Sephadex LH-20 column (Pharmacia Biotech, Uppsala, Sweden). Methanol (50% dissolved in water) and 70% acetone (dissolved in water) were used to elute successionally. The latter was freeze-dried to get dry proanthocyanidin powders after the acetone was removed by rotary evaporation. These powders were stored in the refrigerator (−20 °C) prior to structure and activity analysis. MALDI-TOF MS Analysis of Hawthorn Fruit Stone Proanthocyanidins. Bruker Reflex III instrument (Bruker-Franzen, Bremen, Germany) was used for MALDI-TOF MS analysis. Samples (proanthocyanidins powders) were irradiated at 337 nm with a nitrogen laser. Angiotensin I (M + H 1296.7, monoisotopic) and angiotensin II (M + H 1046.5, monoisotopic) were employed to calibrate mass spectra. Parameters for positive pattern spectra in the reflectron pattern were as follows: accelerating voltage, 20 kV; reflectron voltage, 23.0 kV; delayed extraction voltage, 16.32 kV; lens voltage, 9.45 kV. Parameters for positive pattern spectra in the linear pattern were as follows: accelerating voltage, 20.0 kV; delayed extraction voltage, 16.25 kV; lens voltage, 10.0 kV. DHB was used

DPPH% inhibition = [(A1 − A 2 )/A1] × 100 124

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where A1 is the absorbance in the absence of the sample and A2 represents the absorbance in the presence of the sample. The IC50 value (μg/mL) is the sample concentration that caused 50% scavenging. ABTS Radical Scavenging Activity Assay. ABTS radical scavenging activity was conducted according to the method of Re et al.20 ABTS solution (7 mM) and potassium persulfate solution (2.45 mM) were mixed and stored in the dark for 16 h to obtain the ABTS+ solution. It was diluted with 80% ethanol to get the ABTS+ working solution. The working solution was detected at 734 nm with an absorbance of 0.700 ± 0.050. Tested sample (0.1 mL) was then mixed with 3.9 mL of ABTS+ working solution. The absorbance was detected on a Beckman UD-800 spectrophotometer (Beckman coulter, Pasadena, California, USA) after incubation for 6 min. The result was expressed as IC50 value. Ferric Reducing Antioxidant Power (FRAP) Assay. The FRAP assay was carried out in compliance with the method described by Benzie and Strain.21 TPTZ solution (10 mM), ferric chloride solution (20 mM), and sodium acetate buffer (300 mM, pH 3.6) were mixed in a ratio of 1:1:10 to get the FRAP working solution. Sample (0.1 mL) was then added into 3 mL of freshly made working solution. The wavelength for the detection was 593 nm. Reaction mixture was kept at room temperature for 10 min before the detection. FRAP values were expressed as millimoles of ascorbic acid equivalents (AAE) per gram of purified proanthocyanidins. Oxygen Radical Absorbance Capacity (ORAC) Assay. The ORAC method was performed on the basis of Ou et al.22 The reaction system (300 μL) included 100 μL (0.33 μM) of fluorescein, 50 μL of extract or Trolox, and 150 μL of AAPH (60 mM). They were shaken well and recorded on a POLARstar Omega (BMG Labtech, Offenburg, Germany) with a 96-well microplate. The detection was performed after that the mixture was kept in 37 °C for 20 min. ORAC value was calculated as micromoles of trolox equivalents per gram of proanthocyanidins (dry weight). Statistical Analysis. All the enzyme assays were repeated three times. The curves present in the picture were made according to the average value. The data for the antioxidant assay were obtained from three independent determinations, and they were analyzed by one-way analysis of variance. It indicated the significant differences when p < 0.05. The software used for the statistical analyses is SPSS 13.0.

Figure 2. MALDI-TOF mass spectra of Chinese hawthorn fruit stone proanthocyanidins. 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. Reflectron MALDI-TOF MS spectra of proanthocyanidins extracted from fruit stone of Chinese hawthorn are displayed in Figure 2A,B. Results indicated that hawthorn fruit stone proanthocyanidins exhibit mass spectra with a primary set of peaks with differences of 288 Da. It can be concluded that 288 Da is a mass difference of one catechin/epicatechin. A subset mass 14 Da higher was also found in the spectra (Figure 2A) except the detected mass series mentioned above. They were produced by gallocatechin/ epigallocatechin. Additionally, the A-type interflavan linkages of 2 Da less than B-type were also detected in the main set of peaks (Figure 2B). These findings suggested that proanthocyanidins extracted from fruit stone of Chinese hawthorn are composed of procyanidin and prodelphindin. B-type procyanidin dominates. These compounds possess structural heterogeneity in monomer units and interflavan linkage (A-type and B-type). Besides the series of peaks described above, 152 Da mass distances following the main set of peaks were also observed (Figure 2A), which are in line with the addition of one galloyl group at the heterocyclic C-ring.23 Also, 132 Da mass distances following the main set of peaks were produced by synchronous attachment of two Cs+ and absence of a proton [M + 2Cs+ − H]+.24 Additionally, these compounds had polymer chain length varying from 2-mers to 12-mers, with most intensity in 4-mers.

To detect polymeric proanthocyanidins higher than those shown above, it is necessary to operate the linear pattern MALDI-TOF MS analysis. Figure 2C reveals a series of peaks separated by 288 Da, which are in keeping with the mass difference of one catechin/epicatechin. These compouds had polymer chain length ranging from monomer to 17-mers. In comparison with spectrum obtained from reflectron pattern (Figure 2A), linear pattern (Figure 2C) had weak resolution, but gave detection of mass information in line with higher DP. Linear MALDI-TOF MS analysis revealed the detection of litchi seed proanthocyanidins up to 27-mers.16 In addition, in our study, proanthocyanidins showed galloyl ester units up to 6-mers in the reflectron pattern, while up to 17-mers in the linear pattern. Reversed-Phase HPLC-ESI-MS Analyses. Chromatogram of thiolytic degraded proanthocyanidins from fruit stone of Chinese hawthorn is shown in Figure 3A. Results indicated that terminal units are mainly epicatechin (peak 2) and the extension units are primarily epicatechin benzylthioethers (peak 8). In addition, catechin (peak 1) and its benzylthioether (peak 7), catechin gallate (peak 3), and epigallocatechin benzylthioether (peak 5) were detected in Chinese hawthorn fruit stone proanthocyanidins by the thiolysis-HPLC method. Because A-type linkage is stable in the process of thiolytic degradation, the A-type terminal unit would be present in the 125



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higher than 10-mers could not be separated and showed a single broad peak at the end of the chromatograms (55.7 min). Our results were similar to the results of Gu et al.26 and a good supplement of the reversed phase HPLC-ESI-MS analysis. Effect of Chinese Hawthorn Fruit Stone Proanthocyanidins on the Monophenolase Reaction of Mushroom Tyrosinase. Kinetic courses of L-tyrosine oxidation by mushroom tyrosinase were tested in the presence of different concentrations of hawthorn proanthocyanidins. When Ltyrosine was used as substrate, the monophenolase activity of tyrosinase was assayed in the absence of inhibitor, and it was found that there was a remarkable lag time. The system reached steady-state rate after the lag period (lag time).27 The steadystate rate of monophenolase activity can be negatively affected by addition of proanthocyanidins from fruit stone of Chinese hawthorn. The steady-state rate decreased in a dose-dependent way following the increase of the inhibitor concentration. When the final concentration of the proanthocyanidins in the system is 166.67 μg/mL, the steady-state rate decreased from 100% to 37.5%. The IC50 value was estimated as 37 ± 0.5 μg/mL for monophenolase activity. However, the corresponding lag time decreased from 94.8 s of the initial state to 47.6 s in the presence of 166.67 μg/mL proanthocyanidins. In our previous study, the samples lengthened the lag time of the enzyme reaction or had no influence on it.28 In the present study, however, proanthocyanidins isolated from fruit stone of Chinese hawthorn accelerated the reaction process (decreased the lag time) of the enzyme. To explain this phenomenon, 3,4dihydroxyphenylalanine, catechin, and epicatechin in different concentrations were added into the enzyme−tyrosine reaction systems (3 mL), and the result showed that all the three compounds can obviously decrease the lag time at a low concentrations. The initial lag time decreased from 277.0 to 140.5, 14.4, and 9.6 s in the presence of 10 μM 3,4dihydroxyphenylalanine, catechin, and epicatechin, respectively. The results showed that o-diphenol could affect the lag period of the monophenolase activity. The presence of o-diphenol in the monophenolase reaction system shortens the lag time, because the lag time is the time required to reach the steady odiphenol concentration.29 These findings further indicated that catechin and epicatechin existing in the extract of fruit stone of Chinese hawthorn are responsible for the particular phenomenon, and the effects of catechin/epicatechin are obviously better than that of 3,4-dihydroxyphenylalanine, which is commonly the substrate of the diphenolase reaction, to shorten the lag period. In summary, proanthocyanidins inhibit monophenolase activity of tyrosinase by reducing its steady-state rate. Effect of Chinese Hawthorn Fruit Stone Proanthocyanidins on the Diphenolase Reaction of Mushroom Tyrosinase. The substrate for the assay of the diphenolase reaction was 3,4-dihydroxyphenylalanine. The relative activity increased before reaching a maximum (124.6% at 40 μg/mL) and decreased after that as concentration of the samples increased (Figure 4A). When the concentration reached 400 μg/mL, the relative activity decreased to 83.3%. Gowda et al.30 found that diphenol can activate diphenolase activity of polyphenol oxidase obtained from field bean (Dolichos lablab). Proanthocyanidins isolated from fruit stone of Chinese hawthorn increased diphenolase reaction absorption of tyrosinase in low concentration. To explain the phenomenon, 3,4-dihydroxyphenylalanine was replaced by catechin/ epicatechin in 3 mL reaction system. The results indicated that both catechin and epicatechin can be better catalyzed by

Figure 3. (A) Reversed-phase HPLC-ESI-MS chromatogram of Chinese hawthorn fruit stone proanthocyanidins after thiolytic degradation. Peaks are as follows: 1, catechin (289 Da, [M − H]−); 2, epicatechin (289 Da, [M − H]−); 3, epicatechin gallate (441 Da, [M − H]−); 4, A-type dimer (575 Da, [M − H]−); 5, epigallocatechin benzylthioether (427 Da, [M − H]−); 6, A-type trimer benzylthioether (985 Da, [M − H]−); 7, catechin benzylthioether (411 Da, [M − H]−); 8, epicatechin benzylthioether (411 Da, [M − H]−); 9, A-type dimer benzylthioether (697 Da, [M − H]−); and 10, benzyl mercaptan. (B) Normal-phase HPLC-ESI-MS chromatogram of Chinese hawthorn fruit stone proanthocyanidins. Peaks are as follows: 1, epicatechin (289 Da [M − H]−); 2, catechin (289 Da [M − H]−); 2a, A-type dimer (575 Da [M − H]−); 3a, A-type trimer (863 Da, [M − H]−); 4a, A-type tetramer (1151 Da, [M − H]−) and 5a, A-type pentamer (1439 Da, [M − H]−), respectively.

form of A-type oligomers, while the A-type linkage between the extension units produced an A-type oligomer benzylthioether.25 Peak 4 was authenticated as A-type dimer. Peaks 6 and 9 were A-type trimer benzylthioether and A-type dimer benzylthioether, respectively. In conclusion, the chromatogram after thiolysis indicated that proanthocyanidins are mainly constituted by the epicatechin unit. In addition, A-type linkage and B-type linkage are both involved in the formation of polymers, with B-type linkage dominating. These results were consistent with the results that achieved by MALDI-TOF MS analysis. Normal-Phase HPLC-ESI-MS Analyses. Normal-phase HPLC profile of proanthocyanidins from fruit stone of Chinese hawthorn is illustrated in Figure 3B. The result indicated that DP of proanthocyanidins range from monomers to the 10mers. In the chromatogram, peaks 1 and 2 are epicatechin (289 Da, [M − H]−) and catechin (289 Da, [M − H]−), respectively. Peaks 2a (575 Da [M − H]−), 3a (863 Da, [M − H]−), 4a (1151 Da, [M − H]−), and 5a (1439 Da, [M − H]−) are peaks of 2-mers, 3-mers, 4-mers, and 5-mers with Atype linkage, respectively. HPLC performance implied the precise separation of proanthocyanidins from monomers to 7mers and the presence of isomers. However, the polymers 126



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Figure 5. Fluorescence emission spectra of mushroom tyrosinase solution in the presence of Chinese hawthorn fruit stone proanthocyanidins with different concentrations. The concentrations of samples for the curves 0−4 are 0, 30, 60, 150, and 300 μg/mL, respectively. The curve A is fluorescence emission spectra of proanthocyanidins at the concentration of 300 μg/mL.

when the concentration of proanthocyanidins increased from 0 to 0.30 mg/mL. In addition, an obvious blue shift is also present in the spectra. The results indicated that when the proanthocyanidins bind to the enzyme molecule, it can induce the enzyme conformation to change and the enzyme activity to decrease. Cu+ Chelating Activity of Proanthocyanidins from Fruit Stone of Chinese Hawthorn. The copper-chelating properties of the proanthocyanidins from fruit stone of Chinese hawthorn were investigated. The increase of Cu(II) concentration to proanthocyanidins solution caused increased absorbance at 280 nm, which shows proanthocyanidins’ binding to Cu(II). There is no absorbance difference at 280 nm when Na2SO4 is added to the proanthocyanidins solution. The inhibitory effect of proanthocyanidins on the tyrosinase may due to the combining capacity between their −OH group and copper ions in the catalytic center of mushroom tyrosinase. Because the −OH groups of proanthocyanidins present anionic form at pH 6.8, they may interact electrostatically with copper ions. Determination of Antioxidant Property. Antioxidant activity of proanthocyanidins isolated from fruit stone of Chinese hawthorn was evaluated by DPPH, ABTS, FRAP, and ORAC assays. Figure 6A,B show DPPH and ABTS radical scavenging activities of proanthocyanidins from fruit stone of Chinese hawthorn and ascorbic acid at different concentrations. The results demonstrated that these compounds could eliminate DPPH and ABTS radicals in a dose-dependent manner. The quality of antioxidants was determined by IC50 values (antioxidant concentration that reduces the DPPH/ ABTS radical by 50%). A much lower value of IC50 indicated better antioxidant activity. The proanthocyanidins had potent DPPH (IC50 170.04 ± 5.17 μg/mL) and ABTS (IC50 122.90 ± 1.25 μg/mL) free radical scavenging activity. One thing special is that proanthocyanidins possessed effective ABTS radical scavenging activity that was significantly better than ascorbic acid (140.52 ± 3.40 μg/mL) (p < 0.05). Noteworthy, the proanthocyanidins from fruit stone of Chinese hawthorn bleached the DPPH and ABTS radical immediately, which suggests that these compounds could be classified as dynamic antioxidants. Next, FRAP assay was used to measure the antioxidant activity of proanthocyanidins, and the same dosedependent activity was found (Figure 6C). The FRAP value was calculated to be 5.83 ± 0.04 mmol AAE/g.

Figure 4. (A) Effects of Chinese hawthorn fruit stone proanthocyanidins on the diphenolase activity of mushroom tyrosinase. [I] represent concentrations of inhibitor. (B) Comparison of diphenolase activity of mushroom tyrosinase after adding 3,4-dihydroxyphenylalanine, catechin, and epicatechin as substrate.

tyrosinase than 3,4-dihydroxyphenylalanine and they are more effective substrates of tyrosinase (Figure 4B). This can be explained because catechin and epicatechin are substrate (3,4dihydroxyphenylalanine) analogues. The increase of relative activity between 0 and 127 μg/mL is mainly because of the high absorption of products produced from catechin/epicatechin (exist in proanthocyanidins of hawthorn fruit stone) and tyrosinase at 475 nm. However, absorptions were not obvious when hawthorn fruit stone proanthocyanidins were mixed with tyrosinase in the absence of 3,4-dihydroxyphenylalanine. It can be speculated that oligomeric proanthocyanidins, such as dimer and trimer, may combine with tyrosinase and produce absorption at 475 nm. These findings suggested that hawthorn proanthocyanidins may inhibit (not activate) the reaction of 3,4-dihydroxyphenylalanine and tyrosinase by competing for the enzyme. Fluorescence Emission Spectra of Mushroom Tyrosinase Solution in the Presence of Chinese Hawthorn Fruit Stone Proanthocyanidins with Different Concentrations. Combining capacities with proanthocyanidins of tyrosinase and its conformational alteration were evaluated by measuring the inherent fluorescence intensity of the enzyme in the absence/presence of proanthocyanidins. The fluorescence emission spectrum is recorded from a range of 300−500 nm with excitation wavelength set at 290 nm. As shown in Figure 5, the tyrosinase produces a strong fluorescence emission (337 nm, curve 0), while proanthocyanidins have a peak at 319 nm (curve A). The concentrations of samples for the curves 1−4 are 30, 60, 150, and 300 μg/mL, respectively. The result showed that fluorescence emission intensity reduced markedly with an increasing concentration of proanthocyanidins. The fluorescence intensity decreased from 64.7 to 13.5 (by 79.4%), 127



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Figure 6. Evaluation of antioxidant activities of Chinese hawthorn fruit stone proanthocyanidins by using (A) DPPH, (B) ABTS, (C) FRAP, and (D) ORAC methods.

Notes

Furthermore, ORAC assay was performed. In the presence of antioxidative compounds, fluorescence decay was inhibited, and fluorescence intensity could be measured at 525 nm with excitation wavelength at 485 nm. The scavenging effect of proanthocyanidins against peroxyl radicals is illustrated in Figure 6D. This effect can be calculated from net integrated area under the kinetic curve and compared with that of Trolox. Our results demonstrated that proanthocyanidins scavenge peroxyl radicals effectively; the ORAC value was 29204.40 ± 870.12 μmol Trolox/g proanthocyanidins, measured at the proanthocyanidins concentration of 0.2 mg/L. The antioxidant activity of proanthocyanidins can be affected by their chemical structure including monomer units, DP, and type of linkage.31 Hagerman et al.32 has conducted insightful research on the mechanism of procyanidin as potential antioxidants, the results of which showed that high molecular weight and hydroxyl group play important roles in free radical scavenging. Therefore, it can be speculated that the procyanidin with high DP in hawthorn fruit stone proanthocyanidins is the reason for the potent activity. Antioxidants have broad application prospects, and researchers have done a great deal of work to search for feasible and effective antioxidants. In this study, our results revealed that proanthocyanidins from fruit stone of Chinese hawthorn may be a good resource for further development as an antioxidant.

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The authors declare no competing financial interest.

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ABBREVIATIONS USED MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; HPLC-ESI-MS, high performance liquid chromatography electrospray ionization mass spectrometry; FRAP, ferric reduced antioxidant power; ORAC, oxygen radical absorbing capacity; ABTS, 2,2-azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt; DPPH, 2,2′-diphenyl-1-picrylhydrazyl; TPTZ, 2,4,6-tripyridylS-triazine; AAPH, 2,20-azobis (2-methylpropionamidine) dihydrochloride

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-592-2185487. Fax: 86-592-2185487. E-mail: chenqx@ xmu.edu.cn. Funding

This study was supported by the Natural Science Foundation of China (Grant Nos. 31071611 and 31070522) and the Science and Technology Foundation of Fujian Province (Grant No. 2010N5013). 128



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Structural Analysis of Proanthocyanidins Isolated from Fruit Stone of

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