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Abstract As a potential source of fine chemicals， unripe apple polyphenols were extracted with Viscozyme L treatment. And then， a preparative moderate pressure liquid chromatography （MPLC） method for separation and purification of polyphenols were successfully established by using silica gel column and octadesyl silane column. The structures of these compounds were identified by 1H and 13C NMR spectroscopy. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction， the yield of apple crude polyphenols （ACP） was 7.08%， and the total phenolic content （TPC） of ACP was 0.95 g GAE/100 g， which were 2 times of that of control; 5 subfractions were obtained from ACP， which were A1， A2， and B1， B2， B3; fractions A2 and B2 were identified as pcoumaric acid and caffeic acid and the yields were 111.54 and 161.70 mg/100 g ACP， respectively. In addition， pcoumaric acid and caffeic acid were the major components in Viscozyme Laided polyphenols extraction， which could be explained by the result of degradation of lignin and chlorogenic acid by carbohydratehydrolyzing enzyme， Viscozyme L.
Key words Unripe apple polyphenole; Viscozyme L; Identification; pcoumaric acid; Caffeic acid
Unripe apples lie scattered about the orchards because of manual thinning out or falling. However， they contain abundant amounts of polyphenol compounds[1-2]that have been reported to exert a variety of biological actions such as free radical scavenging activity[3-4]， antiallergic activity[5-6]， and antiarteriosclerosis activity[7-8]. The main classes of polyphenols in apples are flavan3ols （catechin， epicatechin， and proanthocyanidin）， hydroxycinnamic acids （chlorogenic acid， caffeic acid， pcoumaric acid， and ferulic acid）， dihydrochalcones （phloridzin and phloretin glucoside）， and flavonols （quercetin and rutin）[2，9]. Interestingly， the contents of these polyphenols are strongly dependent on their varieties and growth stage[6]. Especially， total phenolic content of unripe apples are 10 times higher than those of ripe apples[1].
Prior to this study， the utilization of carbohydratehydrolyzing enzymes for the enhancement of polyphenol extraction efficiency from unripe apples[10]， and apple pomace were investigated[11]. In addition， the production of apple antioxidant polyphenol （AAP） from apple crude solution （ACS） was also developed by adsorptiondesorption process using Amberlite XAD series resins[12]. We had also tried to utilize Viscozyme L for enhancing the extraction efficiency of unripe apple polyphenols[13]. However， there have been few relative reports on the purification and identification of polyphenol compounds from unripe apples with carbohydratehydrolyzing enzymesaided extraction. In this study， unripe apple polyphenols were extracted with Viscozyme L treatment and purified by silica gel column and octadesylsilane column， and the structures of major phenolic compounds were identified by 1H and 13C NMR spectroscopy.
Materials and Methods
Materials
Unripe apples （Malus pumila cv. Fuji） were collected 85 d after full bloom from the orchard of Kyungpook National University in Daegu， Korea， in 2015， and stored in a freezer （-70） until the experiment.
Reagents and instruments
Viscozyme L （from Aspergillus aculeatus， 100 fungal betaglucanase units （FBG）/ml， Novozymes A/S， Bagsvaerd， Denmark） was used in this study. Polyphenol standards were obtained from Sigma Co. （St. Louis， MO.）. The organic solvents and chemicals were analytical grade and purchased from Duksan Co. （Seoul， Korea）. Moderate pressure liquid chromatography （MPLC， Yamazen 540， Japan）， and NMR Spectrometer （AVANCE digital 400， Bruker， Germany） were used in this study.
Enrichment of polyphenols
10 kg of whole unripe apples were blanched at 85 for 15 min for the inhibition of polyphenol oxidase （PPO）， then crushed and homogenized with equal volume of water[14]. The homogenized solution was incubated at 47.12， for 12.52 h with adding 1.95% Viscozyme L to obtain unripe apple crude solution （ACS）[10]. Then ACS was filtered and concentrated to unripe apple crude polyphenols （ACP） with a rotary evaporator[12]. The extraction yield of ACP was calculated as the percent ratio of the weight of extract to that of the unripe apple samples. The control test without Viscozyme L treatment was also performed.
Determination of total phenolic content
Total phenolic content （TPC） of ACP was examined using FolinCiocalteus reagent method with some modifications[15]. Quantification was done on the basis of standard curve with gallic acid. TPC is expressed as gallic acid equivalents （GAE） in grams per 100 g fresh weight （g GAE/100 g）.
Isolation and purification unripe apple polyphenols
80 g of ACP with enzymeaided extraction was fractionated according to its polarity as shown in Fig. 1. The ethyl acetate portion was concentrated under vacuum to give 670 mg of residue， which was used for MPLC separation. First， it was chromatographed on a silica gel column （30 mm≠400 mm， i.d.） using CHCl3≥MeOH （100≥0-0≥100） gradient to achieve 100 fractions （1-100）. According to the results of UV absorbance （290 nm） and thin layer chromatography （TLC）， developed by a solvent system of methanol/water （60≥40， v/v）， these fractions were combined to 5 group fractions （Fr. AE）. Fr. A and Fr. B were evaporated to dryness under vacuum for further fractionation to obtain subfractions by an Octadesylsilane column （ODS， 30≠300 mm， i.d.） using stepgradient elution of MeOH≥H2O （0≥100-100≥0） and TLC. The subfractions were identified by 1H NMR and 13C NMR spectra. The yield of each group fraction and subfraction was calculated and expressed as mg/100 g ACP after evaporation and concentration. Identification of unripe apple polyphenols
Purified polyphenols were identified by NMR spectroscopy on a Bruker model AMX400 instrument. Samples （10-20 mg） were dissolved in 0.4 ml of deuterated acetone， methanol or dimethylsulphoxide （DMSO） and chemical shifts were referenced to TMS （1H） or solvent signal （13C）.
Statistical analysis
All examinations were executed in triplicate， and the values are reported as mean÷standard deviation. Statistical analyses were performed by the Statistical Analysis System （SAS version 9.1， SAS Institute Inc. NC， US， 2003）. Values were evaluated by the analysis of variance （ANOVA）， followed by Duncans multiple range tests （P<0.05）.
Results and Analysis
Extraction efficiency of unripe apple polyphenols with Viscozyme L treatment
In order to enhance the extraction of polyphenols from the unripe apples， carbohydratehydrolyzing enzyme （Viscozyme L） aided polyphenol extraction techniques have been studied in this study. ACP extraction yield and TPC of ACP comparisons between Viscozyme Laided extraction condition and control treatments are shown in Table 1. The results indicate that ACP extraction yield （7.08%） and TPC of ACP （0.95 g GAE/100g） increased by about 2.5 and 2 times， respectively， compared with the control treatment， indicating the occurrence of more active hydrolysis reaction under the enzymeaided condition.
Selective isolation of polyphenols
The isolation and identification of unripe apple polyphenols was carried out by means of MPLC， UVVisible Spectrophotometer （290 nm）， and TLC. The silica gel column fraction provided 5 group fractions （Fr. A， 118; B， 1928; C， 2938; D， 3947 and E， 5565） according to the results of UV absorbance （Fig. 2） and TLC （data not shown）. The higher amount of fractions Fr. A （335.59 mg/100 g ACP） and Fr. B （287.27 mg/100 g ACP） were further fractionated with ODS column and the elution was isolated to subfractions A1， A2 and B1， B2， B3 （Fig. 1） according to TLC results （data not shown）. The higheryielding pure subfractions were A2 （111.54 mg/100 g ACP） and B2 （161.70 mg/100 g ACP）. Therefore， subfractions A2 and B2 were identified by 1HNMR and 13CNMR analysis.
Identification of polyphenols
Subfraction A2 was identified， and the results are showed in Table 2， Fig. 3 and Fig. 4. The 1H NMR spectroscopic data indicated that presence of two transcoupled vinylic protons at 7.56 （1H， dd， J=15.8 Hz） and 6.32 （1H， d， J=15.8 Hz）. In the aromatic region， two sets of chemically equivalent protons at 7.45 （H2/H6， dd， J=1.8 and 6.8 Hz） and 6.63 （H3/H5， dd， J=1.8 and 6.8 Hz） suggested a 1， 4disubstituted aromatic ring. In addition， the 13C NMR spectrum exhibited two chemically equivalent aromatic carbons at 129.91 （C2/C6） and 115.75 （C3/C5）. Based on these spectroscopic data and comparison with the literature[16-17]， the A2 was identified as pcoumaric acid. Subfraction B2 was identified， and the results are showed in Table 3， Fig. 4 and Fig. 5. B2 constitution was evident from the 1H and 13C NMR spectra which showed the presence of an aromatic ring， an ethylene moiety as well as a carboxylic acid group. The trans configuration of the double bond was shown by the two doublets at 6.18 （1H， d） and 7.42 （1H， d） with a large coupling constant （J=15.8 Hz）. The ABC type coupling pattern of the aromatic protons （ 6.75， 6.97 and 7.03） in the 1H NMR spectrum was evidence of a 1，2，4trisubstituted phenyl ring （Table 3）. This was corroborated by 13C NMR data which were consistent with a catechol aromatic ring hence B2 was identified as caffeic acid[17-19].
Conclusions and Discussion
The use of carbohydratehydrolyzing enzymes， such as pectinase， cellulase， hemicellulase， and glucanase has been recently introduced to release cell wall complex polyphenol from plant materials[20]. These enzymes play a role in disintegrating the plant cell wall matrix and facilitating the polyphenol extraction[12]. Based on the enzymatic pretreatment， Viscozyme L was selected to extraction polyphenols from unripe apples. Our results indicated apple cell wall was decomposed by Viscozyme L and released polyphenols， thereby improving total phenolic content and the polyphenol extraction yield （Table 1）. Furthermore， our results are in accordance with Khanizadeh et al.[21]and Park and Kim[22]， who reported that carbohydratehydrolyzing enzymes significantly enhanced the total phenolic content extraction from the apple pomace and peel at the dosage of 2%-10%.
The apple cell wall is a complex network that is composed of polysaccharides， such as cellulose， hemicelluloses， pectin， and lignin[23]. Especially， lignin is the major phenolic constituent， deposited together with hydroxycinnamic acids （pcoumaric and ferulic acid）， simple flavonoids， and procyanidin B2[24]. Hence， pcoumaric acid has been recognized as an indicator of decomposition of lignin during the enzyme hydrolysis process[13]. Furthermore， chlorogenic acid （3 or 5caffeoylquinic acid） is the ester compound of caffeic acid and quinic acid and was the major phenolic compound of apples in reported by other researchers. However， in this study， pcoumaric acid and caffeic acid were the major component in enzymeaided polyphenols extraction， which could be explained by the result of degradation of lignin and chlorogenic acid by enzyme， Viscozyme L[13].
pcoumaric acid and caffeic acid are phenylpropanoids playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. These two compounds are also an antioxidant and have potential utility as a general protectant against free radicals. In this study， polyphenols extracted from unripe apples with Viscozyme L extraction could be successfully separated by column chromatography on a silica gel column and octadesylsilane column which resulted in the isolation of A1， A2， B1， B2 and B3. And then， A2 and B2 were identified as pcoumaric acid and caffeic acid， respectively. In addition， the experiment yield of pcoumaric acid and caffeic acid was 111.54 and 161.70 mg per 100 g ACP， respectively. The result showed that ACP contained a high level of polyphenols， pcoumaric acid and caffeic acid， which could be commercially exploited. References
[1]ZHENG HZ， KIM YL， CHUNG SK. A profile of physicochemical and antioxidant changes during fruit growth for the utilisation of unripe apples[J]. Food Chemistry， 2012，131： 106-110.
[2]SCHIEBER A， PETRA K， REINHOLD C. Determination of phenolic acids and flavonoids of apple and pear by highperformance liquid chromatography[J]. Journal of Chromatography A， 2001， 910： 265-273.
[3]ADIL ?H， ??ETIN H?， YENER ME， et al. Subcritical （carbon dioxide+ethanol） extraction of polyphenol from apple and peach pomaces， and determination of the antioxidant activities of the extracts[J]. Journal of supercritical fluids， 2007， 43（1）： 55-63.
[4]SUDHA ML， BASKARAN V， LEELAVATHI K. Apple pomace as a source of dietary fiber and polyphenol and its effect on the rheological characteristics and cake making[J]. Food Chemistry， 2007， 104（2）： 686-692.
[5]KOJIMA T， AKIYAMA S， TANIUCHI S， et al. Antiallergic effect of apple polyphenol on patients with atopic dermatitis： a pilot study[J]. Allergology International， 2000， 49： 69-73.
[6]AKIYAMA H， YUJI S， TAKAHIRO W， et al. Dietary unripe apple polyphenol inhibits the development of food allergies in murine models[J]. FEBS Letters， 2005， 579： 4485-4491.
[7]DANGELO S， CIMMINO A， RAIMO M， et al. Effect of reddeningripening on the antioxidant activity of polyphenol extracts from Cv. ‘Annurca’ apple fruits[J]. Journal of Agricultural and Food Chemistry， 2007， 55（24）： 9977-9985.
[8]STEFANIA DA， AMELIA C， MARIANNA R， et al. Effect of reddeningripening on the antioxidant activity of polyphenol extracts from Cv. ‘Annurca’ apple fruits[J]. Journal of Agricultural and Food Chemistry， 2007， 55（24）： 9977-9985.
[9]ALONSOSALCES RM， HERRERO C， BARRANCO A. Classification of apple fruits according to their maturity state by the pattern recognition analysis of their polyphenolic compositions[J]. Food Chemistry， 2005， 93（1）：113-123.
[10]ZHENG HZ， HWANG IW， KIM SK， et al. Optimization of Carbohydratehydrolyzing Enzyme Aided Polyphenol Extraction from Unripe Apples[J]. Journal of the Korean Society for Applied Biological Chemistry， 2010， 53（3）： 342-350.
[11]ZHENG HZ， LEE HR， LEE SH， et al. Pectinase assisted extraction of polyphenol from apple pomace[J]. Chinese Journal of Analytical Chemistry， 2008， 36（3）： 306-310.
[12]ZHENG HZ， HWANG IW， KIM BK， et al. Phenolics Enrichment Process from Unripe Apples[J]. Journal of the Korean Society for Applied Biological Chemistry， 2014， 57（4）：457-461. [13]ZHENG HZ， HWANG IW， CHUNG SK. Enhancing polyphenol extraction from unripe apples by carbohydratehydrolyzing enzymes[J]. Journal of Zhejiang UniversitySCIENCE B， 2009， 10（12）： 912-919.
[14]BUCKOW R， WEISS U， DIETRICH K. Inactivation kinetics of apple polyphenol oxidase in different pressuretemperature domains[J]. Innovative food science & emerging technologies， 2009， 10（4）： 441-448.
[15]SINGLETON VL， ORTHOFER R， LAMUELARAVENTOS RM. Analysis of total phenolic and other oxidation substrates and antioxidants by means of FolinCiocalteu reagent[J]. Methods in Enzymology， 1999， 299： 152-178.
[16]BERGMAN M， LUCY V， HUGO EG， et al. The antioxidant activity of aqueous spinach extract： chemical identification of active fractions[J]. Phytochemistry， 2001， 58： 143-152.
[17]NEDIME D， SE?KIN ?， ESRA U， et al. The isolation of carboxylic acids from the flowers of Delphinium formosum[J]. Turkish Journal Of Chemistry， 2001， 25： 93-97.
[18]LU YR， FOO LY. Identification and quantification of major polyphenols in apple pomace[J]. Food Chemistry， 1997， 59（2）： 187-194.
[19]LIM EK， GILLIAN SH， LI Y， et al. Regioselectivity of glucosylation of caffeic acid by a UDPglucose： glucosyltransferase is maintained in planta[J]. Biochemical Journal， 2003， 373： 987-992.
[20]S?RENSEN HR， PEDERSEN S， ANDERS VN， et al. Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue[J]. Enzyme and Microbial Technology， 2005， 36（5-6）： 773-784.
[21]KHANIZADEH S， TSAO R， REKIKA D， et al. Polyphenol composition and total antioxidant capacity of selected apple genotypes for processing[J]. Journal of food composition and analysis， 2008， 21（5）： 396-401.
[22]PARK MK， KIM CH. Extraction of polyphenols from apple peel using cellulase and pectinase and estimation of antioxidant activity[J]. Journal of the Korean Society of Food Science and Nutrition， 2009， 38（5）： 535-540 （in Korea）.
[23]LE BOURVELLEC C， GUYOT S， RENARD CMGC. Interactions between apple （Malus x domestica Borkh.） polyphenol and cell walls modulate the extractability of polysaccharides[J]. Carbohydrate Polymers， 2009， 75（2）： 251-261.
[24]PINELO M， ZORNOZA B， MEYER AS. Selective release of phenols from apple skin： mass transfer kinetics during solvent and enzymeassisted extraction[J]. Separation and Purification Technology， 2008， 63（3）： 620-627.
Key words Unripe apple polyphenole; Viscozyme L; Identification; pcoumaric acid; Caffeic acid
Unripe apples lie scattered about the orchards because of manual thinning out or falling. However， they contain abundant amounts of polyphenol compounds[1-2]that have been reported to exert a variety of biological actions such as free radical scavenging activity[3-4]， antiallergic activity[5-6]， and antiarteriosclerosis activity[7-8]. The main classes of polyphenols in apples are flavan3ols （catechin， epicatechin， and proanthocyanidin）， hydroxycinnamic acids （chlorogenic acid， caffeic acid， pcoumaric acid， and ferulic acid）， dihydrochalcones （phloridzin and phloretin glucoside）， and flavonols （quercetin and rutin）[2，9]. Interestingly， the contents of these polyphenols are strongly dependent on their varieties and growth stage[6]. Especially， total phenolic content of unripe apples are 10 times higher than those of ripe apples[1].
Prior to this study， the utilization of carbohydratehydrolyzing enzymes for the enhancement of polyphenol extraction efficiency from unripe apples[10]， and apple pomace were investigated[11]. In addition， the production of apple antioxidant polyphenol （AAP） from apple crude solution （ACS） was also developed by adsorptiondesorption process using Amberlite XAD series resins[12]. We had also tried to utilize Viscozyme L for enhancing the extraction efficiency of unripe apple polyphenols[13]. However， there have been few relative reports on the purification and identification of polyphenol compounds from unripe apples with carbohydratehydrolyzing enzymesaided extraction. In this study， unripe apple polyphenols were extracted with Viscozyme L treatment and purified by silica gel column and octadesylsilane column， and the structures of major phenolic compounds were identified by 1H and 13C NMR spectroscopy.
Materials and Methods
Materials
Unripe apples （Malus pumila cv. Fuji） were collected 85 d after full bloom from the orchard of Kyungpook National University in Daegu， Korea， in 2015， and stored in a freezer （-70） until the experiment.
Reagents and instruments
Viscozyme L （from Aspergillus aculeatus， 100 fungal betaglucanase units （FBG）/ml， Novozymes A/S， Bagsvaerd， Denmark） was used in this study. Polyphenol standards were obtained from Sigma Co. （St. Louis， MO.）. The organic solvents and chemicals were analytical grade and purchased from Duksan Co. （Seoul， Korea）. Moderate pressure liquid chromatography （MPLC， Yamazen 540， Japan）， and NMR Spectrometer （AVANCE digital 400， Bruker， Germany） were used in this study.
Enrichment of polyphenols
10 kg of whole unripe apples were blanched at 85 for 15 min for the inhibition of polyphenol oxidase （PPO）， then crushed and homogenized with equal volume of water[14]. The homogenized solution was incubated at 47.12， for 12.52 h with adding 1.95% Viscozyme L to obtain unripe apple crude solution （ACS）[10]. Then ACS was filtered and concentrated to unripe apple crude polyphenols （ACP） with a rotary evaporator[12]. The extraction yield of ACP was calculated as the percent ratio of the weight of extract to that of the unripe apple samples. The control test without Viscozyme L treatment was also performed.
Determination of total phenolic content
Total phenolic content （TPC） of ACP was examined using FolinCiocalteus reagent method with some modifications[15]. Quantification was done on the basis of standard curve with gallic acid. TPC is expressed as gallic acid equivalents （GAE） in grams per 100 g fresh weight （g GAE/100 g）.
Isolation and purification unripe apple polyphenols
80 g of ACP with enzymeaided extraction was fractionated according to its polarity as shown in Fig. 1. The ethyl acetate portion was concentrated under vacuum to give 670 mg of residue， which was used for MPLC separation. First， it was chromatographed on a silica gel column （30 mm≠400 mm， i.d.） using CHCl3≥MeOH （100≥0-0≥100） gradient to achieve 100 fractions （1-100）. According to the results of UV absorbance （290 nm） and thin layer chromatography （TLC）， developed by a solvent system of methanol/water （60≥40， v/v）， these fractions were combined to 5 group fractions （Fr. AE）. Fr. A and Fr. B were evaporated to dryness under vacuum for further fractionation to obtain subfractions by an Octadesylsilane column （ODS， 30≠300 mm， i.d.） using stepgradient elution of MeOH≥H2O （0≥100-100≥0） and TLC. The subfractions were identified by 1H NMR and 13C NMR spectra. The yield of each group fraction and subfraction was calculated and expressed as mg/100 g ACP after evaporation and concentration. Identification of unripe apple polyphenols
Purified polyphenols were identified by NMR spectroscopy on a Bruker model AMX400 instrument. Samples （10-20 mg） were dissolved in 0.4 ml of deuterated acetone， methanol or dimethylsulphoxide （DMSO） and chemical shifts were referenced to TMS （1H） or solvent signal （13C）.
Statistical analysis
All examinations were executed in triplicate， and the values are reported as mean÷standard deviation. Statistical analyses were performed by the Statistical Analysis System （SAS version 9.1， SAS Institute Inc. NC， US， 2003）. Values were evaluated by the analysis of variance （ANOVA）， followed by Duncans multiple range tests （P<0.05）.
Results and Analysis
Extraction efficiency of unripe apple polyphenols with Viscozyme L treatment
In order to enhance the extraction of polyphenols from the unripe apples， carbohydratehydrolyzing enzyme （Viscozyme L） aided polyphenol extraction techniques have been studied in this study. ACP extraction yield and TPC of ACP comparisons between Viscozyme Laided extraction condition and control treatments are shown in Table 1. The results indicate that ACP extraction yield （7.08%） and TPC of ACP （0.95 g GAE/100g） increased by about 2.5 and 2 times， respectively， compared with the control treatment， indicating the occurrence of more active hydrolysis reaction under the enzymeaided condition.
Selective isolation of polyphenols
The isolation and identification of unripe apple polyphenols was carried out by means of MPLC， UVVisible Spectrophotometer （290 nm）， and TLC. The silica gel column fraction provided 5 group fractions （Fr. A， 118; B， 1928; C， 2938; D， 3947 and E， 5565） according to the results of UV absorbance （Fig. 2） and TLC （data not shown）. The higher amount of fractions Fr. A （335.59 mg/100 g ACP） and Fr. B （287.27 mg/100 g ACP） were further fractionated with ODS column and the elution was isolated to subfractions A1， A2 and B1， B2， B3 （Fig. 1） according to TLC results （data not shown）. The higheryielding pure subfractions were A2 （111.54 mg/100 g ACP） and B2 （161.70 mg/100 g ACP）. Therefore， subfractions A2 and B2 were identified by 1HNMR and 13CNMR analysis.
Identification of polyphenols
Subfraction A2 was identified， and the results are showed in Table 2， Fig. 3 and Fig. 4. The 1H NMR spectroscopic data indicated that presence of two transcoupled vinylic protons at 7.56 （1H， dd， J=15.8 Hz） and 6.32 （1H， d， J=15.8 Hz）. In the aromatic region， two sets of chemically equivalent protons at 7.45 （H2/H6， dd， J=1.8 and 6.8 Hz） and 6.63 （H3/H5， dd， J=1.8 and 6.8 Hz） suggested a 1， 4disubstituted aromatic ring. In addition， the 13C NMR spectrum exhibited two chemically equivalent aromatic carbons at 129.91 （C2/C6） and 115.75 （C3/C5）. Based on these spectroscopic data and comparison with the literature[16-17]， the A2 was identified as pcoumaric acid. Subfraction B2 was identified， and the results are showed in Table 3， Fig. 4 and Fig. 5. B2 constitution was evident from the 1H and 13C NMR spectra which showed the presence of an aromatic ring， an ethylene moiety as well as a carboxylic acid group. The trans configuration of the double bond was shown by the two doublets at 6.18 （1H， d） and 7.42 （1H， d） with a large coupling constant （J=15.8 Hz）. The ABC type coupling pattern of the aromatic protons （ 6.75， 6.97 and 7.03） in the 1H NMR spectrum was evidence of a 1，2，4trisubstituted phenyl ring （Table 3）. This was corroborated by 13C NMR data which were consistent with a catechol aromatic ring hence B2 was identified as caffeic acid[17-19].
Conclusions and Discussion
The use of carbohydratehydrolyzing enzymes， such as pectinase， cellulase， hemicellulase， and glucanase has been recently introduced to release cell wall complex polyphenol from plant materials[20]. These enzymes play a role in disintegrating the plant cell wall matrix and facilitating the polyphenol extraction[12]. Based on the enzymatic pretreatment， Viscozyme L was selected to extraction polyphenols from unripe apples. Our results indicated apple cell wall was decomposed by Viscozyme L and released polyphenols， thereby improving total phenolic content and the polyphenol extraction yield （Table 1）. Furthermore， our results are in accordance with Khanizadeh et al.[21]and Park and Kim[22]， who reported that carbohydratehydrolyzing enzymes significantly enhanced the total phenolic content extraction from the apple pomace and peel at the dosage of 2%-10%.
The apple cell wall is a complex network that is composed of polysaccharides， such as cellulose， hemicelluloses， pectin， and lignin[23]. Especially， lignin is the major phenolic constituent， deposited together with hydroxycinnamic acids （pcoumaric and ferulic acid）， simple flavonoids， and procyanidin B2[24]. Hence， pcoumaric acid has been recognized as an indicator of decomposition of lignin during the enzyme hydrolysis process[13]. Furthermore， chlorogenic acid （3 or 5caffeoylquinic acid） is the ester compound of caffeic acid and quinic acid and was the major phenolic compound of apples in reported by other researchers. However， in this study， pcoumaric acid and caffeic acid were the major component in enzymeaided polyphenols extraction， which could be explained by the result of degradation of lignin and chlorogenic acid by enzyme， Viscozyme L[13].
pcoumaric acid and caffeic acid are phenylpropanoids playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. These two compounds are also an antioxidant and have potential utility as a general protectant against free radicals. In this study， polyphenols extracted from unripe apples with Viscozyme L extraction could be successfully separated by column chromatography on a silica gel column and octadesylsilane column which resulted in the isolation of A1， A2， B1， B2 and B3. And then， A2 and B2 were identified as pcoumaric acid and caffeic acid， respectively. In addition， the experiment yield of pcoumaric acid and caffeic acid was 111.54 and 161.70 mg per 100 g ACP， respectively. The result showed that ACP contained a high level of polyphenols， pcoumaric acid and caffeic acid， which could be commercially exploited. References
[1]ZHENG HZ， KIM YL， CHUNG SK. A profile of physicochemical and antioxidant changes during fruit growth for the utilisation of unripe apples[J]. Food Chemistry， 2012，131： 106-110.
[2]SCHIEBER A， PETRA K， REINHOLD C. Determination of phenolic acids and flavonoids of apple and pear by highperformance liquid chromatography[J]. Journal of Chromatography A， 2001， 910： 265-273.
[3]ADIL ?H， ??ETIN H?， YENER ME， et al. Subcritical （carbon dioxide+ethanol） extraction of polyphenol from apple and peach pomaces， and determination of the antioxidant activities of the extracts[J]. Journal of supercritical fluids， 2007， 43（1）： 55-63.
[4]SUDHA ML， BASKARAN V， LEELAVATHI K. Apple pomace as a source of dietary fiber and polyphenol and its effect on the rheological characteristics and cake making[J]. Food Chemistry， 2007， 104（2）： 686-692.
[5]KOJIMA T， AKIYAMA S， TANIUCHI S， et al. Antiallergic effect of apple polyphenol on patients with atopic dermatitis： a pilot study[J]. Allergology International， 2000， 49： 69-73.
[6]AKIYAMA H， YUJI S， TAKAHIRO W， et al. Dietary unripe apple polyphenol inhibits the development of food allergies in murine models[J]. FEBS Letters， 2005， 579： 4485-4491.
[7]DANGELO S， CIMMINO A， RAIMO M， et al. Effect of reddeningripening on the antioxidant activity of polyphenol extracts from Cv. ‘Annurca’ apple fruits[J]. Journal of Agricultural and Food Chemistry， 2007， 55（24）： 9977-9985.
[8]STEFANIA DA， AMELIA C， MARIANNA R， et al. Effect of reddeningripening on the antioxidant activity of polyphenol extracts from Cv. ‘Annurca’ apple fruits[J]. Journal of Agricultural and Food Chemistry， 2007， 55（24）： 9977-9985.
[9]ALONSOSALCES RM， HERRERO C， BARRANCO A. Classification of apple fruits according to their maturity state by the pattern recognition analysis of their polyphenolic compositions[J]. Food Chemistry， 2005， 93（1）：113-123.
[10]ZHENG HZ， HWANG IW， KIM SK， et al. Optimization of Carbohydratehydrolyzing Enzyme Aided Polyphenol Extraction from Unripe Apples[J]. Journal of the Korean Society for Applied Biological Chemistry， 2010， 53（3）： 342-350.
[11]ZHENG HZ， LEE HR， LEE SH， et al. Pectinase assisted extraction of polyphenol from apple pomace[J]. Chinese Journal of Analytical Chemistry， 2008， 36（3）： 306-310.
[12]ZHENG HZ， HWANG IW， KIM BK， et al. Phenolics Enrichment Process from Unripe Apples[J]. Journal of the Korean Society for Applied Biological Chemistry， 2014， 57（4）：457-461. [13]ZHENG HZ， HWANG IW， CHUNG SK. Enhancing polyphenol extraction from unripe apples by carbohydratehydrolyzing enzymes[J]. Journal of Zhejiang UniversitySCIENCE B， 2009， 10（12）： 912-919.
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