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Structural features and antioxidant activity of tannin frompersimmon pulp

Hai-Feng Gu a, Chun-Mei Li a,*, Yu-juan Xu a,b, Wan-feng Hu a, Mei-hong Chen a,Qiong-hong Wan a

a College of Food Science and Technology, Hua Zhong Agricultural University, Wuhan City, Hubei Province 430070, Chinab Sericulture and Farm Produce Processing Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou City,

Guangdong Province 510610, China

Received 24 August 2007; accepted 25 November 2007

Abstract

Phenolic compounds from persimmon pulp were extracted with methanol acidified with 1% HCl, and then purified on AB-8 macro-porous resin. The tannic extracts obtained were fractionated by polysulfone ultrafiltration membrane with molecular weight cutoff of10,000 Da into two fractions: low molecular weight tannin (LMWT) and high molecular weight tannin (HMWT). HPLCMS analysisshowed that gallic acid was one of the main components of LMWT fraction. The molecular weight distribution of HMWT was deter-mined to be in the range of 1.16 104 Da to 1.54 104 Da, with the molecular weight of 1.28 104 Da in Mn and 1.39 10

4 Da in Mwby GPC method. HPLCMS showed that the thiolysis degradation products of HMWT consist of (epi) gallocatechin, epigallocatechin-3-O-gallate, epicatechin-3-O-gallate and an unknown monomer with the ratio of 1:7:3:1 by estimation of the peak area on HPLC. Theantioxidant properties of persimmon tannins were evaluated using the hydroxyl radical scavenging activities by 2-deoxyribose oxidation

system and salicylic acid system, superoxide anion scavenging activity, and linoleic acid lipid peroxidation inhibition activity, respec-tively. HMWT exhibited excellent antioxidant activities in all tested systems in a dose-dependent manner. The antioxidant activity ofHMWT was significantly stronger than that of LMWT and grape seeds proanthocyanidins (GSP), suggesting that high molecular weightcondensed tannins are the major antioxidant composition in persimmon pulp. 2007 Elsevier Ltd. All rights reserved.

Keywords: Persimmon (Diospyros kakiL.) pulp; Condensed tannin; Structural features; Antioxidant activity

1. Introduction

Persimmon (Diospyros kaki L.), which belongs to the

Ebenaceae family, is widespread in China, Japan andKorea. In China, its fruits and leaves were traditionallyused for many medicinal purposes such as coughs, hyper-tension, dyspnoea, paralysis, frostbite, burns and bleeding(Mowat, 1990). Persimmon fruits were also reported toexercise hypercholesterolemia, antioxidant and free radical

scavenging effects (Matsuo & Ito, 1978; Shela, Elzbieta,Gustaw, Marina, & Simon, 1998). Persimmon fruit con-tains abundant polyphenols, including condensed tannin

and other phenolic compounds, which are related to thevarious physiological functions, including detoxificationeffect on snake venom as well as toxic substances producedby microorganisms (Okonogi, & Hattori, 1978; Okonogi,Hattori, Ogiso, & Mitsui, 1979) the inhibitory effects onhuman lymphoid leukemia cells (Achiwa, Hibasami,Katsuzaki, Imai, & Komiya, 1997; Hibasami, Achiwa,Fujikawa, & Komiya, 1996); the inhibitory effects on themutagenicity of C-nitro and C-nitroso compounds (Achi-wa, Hibasami, Katsuzaki, Kada, & Komiya, 1996); andthe inhibition of the incidence of stroke and the extension

0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodres.2007.11.011

* Corresponding author. Tel.: +86 13296599657.E-mail addresses:[email protected],[email protected](C.-M.

Li).

www.elsevier.com/locate/foodres

Available online at www.sciencedirect.com

Food Research International 41 (2008) 208217
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of the life span of stroke-prone spontaneously hypertensiverats (Ushida et al., 1995). However, due to the large molec-ular structure and high absorbability, the separation anddetermination of persimmon tannin is difficult and manyof the above research used crude persimmon extract orpersimmon peel and pulp as test materials. The crude plant

extract is a very complex mixture containing sometimeshundreds of different compounds such as p-coumaric, gal-lic acid, catechin, flavonoids and condensed tannin existingin persimmon (Mallavadhani, Panda, & Rao, 1998), butnot all the phenolic compounds from persimmon are ofphysiological concern. One objective of this study is toinvestigate whether high molecular weight condensedtannin is the main antioxidant composition in persimmonpulp.

Furthermore, it is meaningful to study the structuralcharacteristic of persimmon tannin to understand its antiox-idant mechanism, but up to now, very few indication wasgiven about its structural characteristics. OnlyMatsuo and

Ito (1978) identified tannin in a Japanese persimmon andfound that the tannin consisted of catechin, catechin-gallate,gallocatechin, gallocatechin-gallate and an unknown termi-nal residue, and belonged to the proanthocyanidin B groupwith carboncarbon interflavan linkage between the C-4 ofone unit and the C-6 (or the C-8) of another unit (Itoo,1986), and had the molecular weight of 1.38 104 Dain Mw.

The structural characteristic of tannin may vary to a sig-nificant extent due to the different breeds and producingareas of persimmon. To some extent, thiolysis degradationcombined with HPLCMS could be the most useful toolfor characterizing the structure of tannin (Tanaka, Takah-

ashi, Kouno, & Nonaka, 1994). In the present work, mac-roporous resin and ultrafiltration combined with GPC andHPLCMSMS were used to separate, purify and charac-terize tannin from persimmon pulp, and the antioxidantactivities were also evaluated.

2. Materials and methods

2.1. Chemicals

Methanol and acetonitrile were of HPLC grade (Fishers,USA), polyvinyl alcohol standard, catechin, gallic acid, lin-oleic acid, and 2-deoxyribose were purchased from Sigma(USA), all solvents and reagents used in analysis were ofanalytical grade from sinopharm chemical reagent factory(Shanghai, China).

2.2. Plant material and sample preparation

Mature and fully colored fruits of the astringent persim-mon were harvested in late October at the orchard inShanxi (China). After harvest, fruits were boiled in fivevolumes of distilled water at 100 C for about 10 min toinactivate polyphenol oxidase. The persimmons werepeeled and the pulp was stored deep frozen at 20 C for

further use.

The pulp were cut into small pieces with a stainless knifeand 50 g of the pulp were extracted with 600 ml methanolacidified with 1% HCl under reflux conditions at 90Cfor 30 min. After filtration, the methanol solution was col-lected. The procedure was repeated two more times and thesolution was combined. The content of total polyphenols in

the combined solution were measured by FolinDenismethod (Gahler, Otto, & Bohm, 2003). Briefly, 1 ml ofextracts and 3 ml of FolinDenis reagent were mixed. After5 min, 3 ml of 10% NaCO3were added and the mixture wasleft at room temperature for 60 min. The absorbance wasmeasured with a spectrophotometer (Shima-dzu UV-2200) at 760 nm. The determination was performed threetimes. The amount of phenolics (expressed as mg gallicacid/g fresh weight) was calculated from a standard curve(20100 mg gallic acid/ml) prepared at the same time.The content of condensed tannins in the combined solutionwere vanillinHCl assay (Chavan, Shahidi, & Naczk,2001). Briefly, to 0.21 ml of the extract, 5 ml of 0.5% van-

illin reagent were added; a 5 ml of 4% concentrated HCl inmethanol was used as a blank. The absorbances of samplesand blank were read at 500 nm after standing for 20 min atroom temperature. Triplicate analyses were conducted andthe mean values were obtained. The amount of condensedtannin (expressed as mg catechin/g fresh weight) was calcu-lated from a standard curve (1050 mg catechin/ml) pre-pared at the same time.

2.3. Separation and purification

The above methanol extracts were evaporated under

vacuum to remove the solvent. The concentrated extractsolution was applied onto a glass column (20 400 mm,i.d.), packed with AB-8 macroporous resin (chemical plantof Nankai University, Tianjin, China), washed firstly withdeionized water at a flow rate of 3 ml/min to remove sugarand other soluble impurity. When practically no more col-ored compounds and sugar were eluted and detected in theoutflow, anhydrous methanol was used to elute the targettannins at a flow rate of 2 ml/min. After eluting, solventwas removed using a rotary evaporator under vacuum,and the residue was lyophilized. The contents of total poly-phenols and condensed tannins in the tannin extract weremeasured by FolinDenis method and vanillinHCl assay,respectively.

Three grams of the above tannin extract was dilutedwith 3000 ml deionized water and then added onto thelayer of polysulfone hollow fiber ultrafine membrane withmolecular weight cutoff of 10,000 Da (Tianjin, Tianfang,China) to remove small molecules. With the operationpressure of 0.05 MPa and filtration flux of 10 L/h, theextract was separated into two parts; low molecular weighttannin (LMWT) in the filtrate and high molecular weighttannin (HMWT) stayed in the retentate. The obtained frac-tions were evaporated and lyophilized for GPC, HPLC,HPLCESIMS analysis and further antioxidant activity

evaluation. Total polyphenols and condensed tannin

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contents of the two fractions were also detected aspreviously.

2.4. HPLC analysis of different fractions of tannic extracts

from persimmon pulp and HPLCESIMS for low

molecular fraction

The separation of low molecules fraction was performedusing a HPLC series 1100 (Hewlett Packard, Waldbronn,Germany) equipped with Chem.-Station software, a degas-ser G1322A, a binary gradient pump G1312A, a thermoautosampler G1329/1330A, and a diode array detectorG1315A. The column used was a 5lm Hypersil ODS2C18 (4.60 mm 200 mm, i.d. Dalian Elite, China). Themobile phase consisted of 2% acetic acid in water, eluentA and acetonitrile, eluent B, at a flow rate of 0.8 ml/min.The gradient program was as follows: 010 min, 010%B,1015 min, 20%B, and 1530 min, 55%B. The injection vol-ume for samples was 10ll, monitoring was performed at

280 nm with the column oven set at 30 C, all samples werefiltered through a 0.45lm syringe filter before analysis.

HPLCESIMS analysis were performed on a Agilent1100 LCMS spectrometer (USA), the chromatographicconditions were the same as described above except thatcolumn: ZORBAX SC- C18(2.1 mm 150 mm, 5lm, Agi-lent, USA) and flow rate: 0.2 ml/min. A source tempera-ture of 500 C, negative ion mode was used with asprayer needle voltage of 3.5 kV. The capillary temperaturewas 350 C. The full scan mass spectra of the tannins fromm/z 100 to 700 were measured using 500 ms for collectiontime and three micro scans were summed.

2.5. Gel permeation chromatography (GPC) and molecular

weight

GPC analysis of HMWT were carried out on AglientHPLC series 1100 at room temperature on an Aglient plaquagel OH 30 column (7.5 mm 300 mm, i.d., Aglient,USA), particle size 8lm. One milligram of HMWT wasdissolved in 1 ml of deionized water, and 50ll was injectedto the column. Water was used as eluent at a flow rate of1 ml/min with the column oven set at 25 C. The eluentwas monitored at 280 nm. Polyvinyl alcohol standard withmolecular weight of 106, 194, 620, 1470, 4120, 11,840,26,000, respectively were used as standard markers forthe molecular weight. Mw and Mn express weight averagemolecular weight and number average molecular weight,respectively (Matsuo & Ito, 1978).

2.6. Thiolysis degradation

One milligram of HMWT was dissolved in 1 ml of meth-anol, and 1 ml of thiolysis reagent (5% solution of benzylmercaptan in methanol containing 0.2 M HCl) was added(Jean, Veronique, Franck, & Michel, 1996; Lingamallu,Hiroshi, Hiroshi, Mayumi, & Mitsuru, 2004). After sealing,

the mixture was shaken and heated at 60 C for 2 h. The

products were then analyzed by HPLC under the followingconditions: solvent A, 0.5% acetic acid; solvent B,methanol, elution with linear gradient: 05 min, 4060%B, 515 min, 80%B, 1530 min, 85%B, the other condi-tions were the same as used in Section2.4for low moleculesanalysis.

2.7. Acid hydrolysis

Water solution and methanol solution of 2 mg/mlHMWT were heated at 80 C for 5 h in 2 M HCl. Thesetwo hydrolyzed products were firstly analyzed by HPLCunder the following conditions: solvent A, 0.5% acetic acidin water; and solvent B, methanol, and the elution gradientconsisted of: 020 min, 525%B, 2030 min, 25%B. Theother conditions were the same as used in Section2.4. Sec-ondly, these two hydrolyzed solutions were analyzed foranthocyanidins by another HPLC conditions as follows:solvent A, 10% formic acid; solvent B, methanolwater

formic acid (45:45:10 v/v/v), elution with linear gradientfrom 35% to 95% B in 20 min, detection at 540 nm, theother conditions were not changed.

All acid hydrolysis solutions were scanned with Phar-macspec UV-1700 spectrophotometer (Shimadzu, Japan)from 800 to 230 nm, compared with the solution beforeacid hydrolysis. All above products were analyzed byHPLCESIMS as the method described in Section 2.4,but for the anthocyanidin it was changed to positive ionmode and the capillary temperature was at 325 C.

2.8. Hydroxyl radical scavenging activity

2.8.1. Hydroxyl radical scavenging activity in 2-deoxyribose

system

The hydroxyl radical scavenging activity of the two frac-tions LMWT and HMWT were assayed by using the 2-deoxyribose oxidation method (Halliwell, 1987) with slightmodification. The reaction mixture contained 0.4 ml of0.2 M sodium phosphate buffer (pH 7.4), 0.1 ml samplesolution of different concentrations, 0.1 ml of 1 mMEDTA, 0.1 ml of 1 mM FeCl3, 0.1 ml of 12 mM hydrogenperoxide, 0.1 ml of 60 mM 2-deoxyribose, and 0.1 ml ofascorbic acid in a tube. After incubation at 37 C for 1 h,the reaction was stopped by adding 1 ml of 2% trichloro-acetic acid and 1 ml of 1.0% thiobarbituric acid. The mix-ture was boiled for 15 min, cooled in ice and extracted byn-butanol and the absorbance was measured at 532 nmagainstn-butanol (as blank). The reaction mixture withouttest sample was used as control, and grape seeds proanth-ocyanidins (GSP) was used as positive control. All theanalyses were done in triplicates and average values weretaken. Hydroxyl radical scavenging ability was evaluatedas the inhibition rate (IR) of 2-deoxyribose oxidation byhydroxyl radical, and expressed as follows:

IR% = [A0(A1 A2)]/A0 100%, where A1, A0, andA2 are the absorbance of the sample, the blank sample,

and the blank control, respectively.

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2.8.2. Hydroxyl radical scavenging activity in salicylic acid

system

The effect of hydroxyl radical scavenging of the fractionLMWT and HMWT were also assayed using the salicylicacid system (Smirnoff & Cumbes, 1996) with slight modifi-cation. Firstly, 1 ml of sample solutions with different con-

centrations, 0.3 ml of 8 mM FeSO4and 0.25 ml of 20 mMhydrogen peroxide were added into the centrifugal vial.Then, 1 ml of 3 mM salicylic acid was put into the test tubeand incubated at 37 C for 30 min. Distilled water (0.45 ml)was added to each vial to make a final volume of 3 ml.After centrifugation (2000 rpm, 10 min), the suspensionswere measured at 510 nm. The reaction mixture withouttest sample was used as control, and grape seeds proanth-ocyanidins (GSP) was used as positive control. All theanalyses were done in triplicates and average values weretaken. Hydroxyl radical scavenging ability was evaluatedas the inhibition rate (IR) of salicylic acid oxidation byhydroxyl radical, and expressed as follows:

IR% = [A0(A1 A2)]/A0 100%, where A1, A0, andA2 are the absorbance of the sample, the blank sampleand the blank control, respectively.

2.9. Superoxide anion radical scavenging activity

The superoxide anion scavenging activity of the fractionLMWT and HMWT were measured by the method ofautoxidation of 1,2,3-trihydroxybenzene (Wu, Zhang,Miao, & Zhu, 2005). The reaction mixture consisted of4.5 ml of 50 mM TrisHCl buffer containing 2 mM EDTA(pH 8.2), and 0.1 ml of sample with different concentra-

tions. The mixture solutions were preincubated at 25 Cfor 10 min. The reaction was initiated by the addition of0.2 ml of 5 mM 1,2,3-trihydroxybenzene (dissolved in10 mM HCl). The absorbance at 320 nm was recordedfor 30 s. 10 mM HCl was served as the blank control foradjusting to zero. As control, distilled water replaced thesample solution was considered as the autoxidation rateof 1,2,3-trihydroxybenzene, and GSP was used as positivecontrol. The scavenging activity on superoxide anion(SASA) radicals was expressed as follows:

SASA% = (V0 V1)/V0 100%, where V0 and V1 arethe autoxidation rate of 1,2,3-trihydroxybenzene (DOD/min), and the autoxidation rate of 1,2,3-trihydroxybenzenein sample (DOD/min), respectively.

2.10. Linoleic acid lipid peroxidation inhibition activity

Lipid peroxidation of linoleic acid was measured by themodified method described previously (Kishida, Toku-maru, & Ishitani, 1993). Each reaction mixture consistedof 4.1 ml of 2.5% linoleic acid in ethanol and 10 ml of0.2 M phosphate buffer (pH 7.4), 1 ml of 2.54 103 g/LFeSO4were added as catalyzer. Different amounts of sam-ples were added to the reaction mixture in a centrifugalvial. The vial was placed in an oven at 40 C. After incuba-

tion for 18 h, the reaction was stopped by adding 1 ml of

25% trichloroacetic acid and 1 ml of 0.67% thiobarbituricacid. The mixture was boiled for 15 min, cooled in iceand the mixture were extracted with 4 ml n-butanol. Aftercentrifugation (8000 rpm, 8 min), then-butanol phase wasmeasured at 532 nm. A control was performed with linoleicacid but without the samples, and GSP was used as positive

control.Inhibition% = [A0(A1A2)]/A0 100%, where A1, A0,andA2are the absorbance of the sample, the blank sample,and the blank control, respectively.

3. Results and discussion

3.1. Purification and fractionation of tannic extract from

persimmon pulp

There are a large amount of low molecular weight sub-stances such as sugar, gallic acid, and organic acid in crudepersimmon methanol extract. The removal of these inter-

fering components from the soluble persimmon extract isan important pretreatment step for the analysis of persim-mon tannin. Purification with macroporous adsorptionresin AB-8 is an effective way to remove sugar and otherlow molecular weight polar substances, after purification,the contents of total polyphenols and condensed tanninsin the crude extract reached 91.1% and 82.4% separately.Matsuo and Ito (1978)reported that the molecular weightof persimmon tannin was quite large. Therefore, furtherpurification with an appropriate filter device is desirableto separate low molecular weight phenolic compoundsfrom high molecular weight tannin. In the present work,

ultrafiltration with polysulfone ultrafine membrane withmolecular weight cutoff of 10,000 Da (Tianjin, Tianfang,China) was used to fractionate the partially purified tannicextract of persimmon pulp. The content of condensed tan-nins of the high molecular weight fraction (retentate)reached 93.4% and no condensed tannin was detected inthe low molecular fraction (filtrate). The content of totalpolyphenols in the filtrate was 87.4%.

HPLC analysis of the crude persimmon methanolextract, the filtrate fraction with low molecular weightand the retentate fraction with high molecular weight wereshown in Fig. 1. The HPLC chromatogram of the crudepersimmon methanol extract (Fig. 1a) showed a largehump with many resolved small peaks on it, but after ultra-filtration, a hump without any resolved small peaks on the280 nm chromatographic profile of the retentate wasobserved (Fig. 1c), suggesting that small molecular pheno-lic compounds could be separated effectively from HMWT.

The four main peaks observed in the LMWT fraction(Fig. 1b) were further analyzed by HPLCMS in the nega-tive ion mode. Peak 1 showed an intense molecular ion[MH] at m/z 169, and was identified as gallic acid onthe basis of its retention time (tR), UVvisible and MSspectra, compared with those of the commercial standard.Both peak 2 and peak 3 showed the same molecular ion

[MH]

at m/z 461 and had a maximum absorbance at


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275 nm and the diagnostic character of phenolic com-pounds. Peak 4 showed an intense molecular ion [MH]

at m/z 609 and exhibited two major absorption peaks at275 nm and 320 nm, respectively, which suggested thatpeak 4 could be of flavone compounds. Nevertheless, fur-ther studies are still required in order to confirm the iden-tities of the three peaks.

3.2. GPC analysis of the high molecular weight persimmon

tannin

Persimmon tannin appeared as a single peak with reten-tion time of 5.244 min on the GPC chromatogram (Fig. 2).This result was quite similar to that of Wu and Hwang

(2002) and Yonemori, Matsushima, and Sugiura (1983).

The molecular weight distribution of persimmon tanninwas determined to be in the range of 1.16 104 Da to1.54 104 Da, with the molecular weight of 1.28 104 Da in Mn and 1.39 10

4 Da in Mw, in reference

to standard curve obtained with polyvinyl alcohol. Theregression equation of the standard curve wasy= 2235x+ 25,010, and theR-squared value was 0.9928.

3.3. Thiolysis degradation

The reversed-phase HPLC chromatogram at 280 nm ofthe high molecular weight persimmon tannin was charac-terized by the presence of a broad hump eluting between15 and 28 min. As observed in previous chromatographicstudies on polyphenolics from other fruits such as apple(Bruno et al., 2000; Peng et al., 2001), such a hump in

the 280 nm chromatographic profile could be attributedto polymeric polyphenols. A series of UVvisible spectraregistered on the whole width of the hump showed a singleabsorption band at 278 nm and all these spectra were quitesimilar to that of ()epicatechin or (+)-catechin, thus sug-gesting that the corresponding phenolic molecules pre-sented structural analogies with these flavanols.

HPLC analysis of the reaction media allowed the assayof the major products resulting from the thiolysis degrada-tion of the HMWT fraction. The disappearance of theaforementioned hump in the chromatographic profile afterthioacidolysis suggested that the degradation was com-plete, and five new peaks were detected distinctly (Fig. 3).Peak 5 was identified as benzyl mercaptan by comparisonof the retention time (tR), UVvisible spectra and massspectra with those of the standard.

Identifications of the other four degradation productswere performed on the basis of their UVvisible spectraand HPLCMS/MS spectrometric determinations in thenegative mode.Fig. 4showed the MS2 spectra of the cor-responding molecular ion peaks of the four thiolysis degra-dation products of HMWT. Peak 1 (Fig. 4a), eluting at9.6 min, showed a molecular ion [MH] at m/z 427 witha characteristic MS2 fragment ion [(MPhCH2S)H]

atm/z303, suggesting that it was stereoisomeric (epi)gallo-

catechin benzylthioethers. Peak 2 (Fig. 4b), eluting at

Fig. 1. HPLC chromatogram of crude extracts (a), LMWT (b) andHMWT (c) (detected at 280 nm).

Fig. 2. GPC chromatogram of the high molecular weight persimmontannin (detected at 280 nm).


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10.6 min, showed a molecular ion [MH] atm/z579 with

a characteristic MS2 fragment ion [(MPhCH2S)H]

atm/z455 and a major fragment ion atm/z303, which cor-responds to the further loss of galloyl residue([(MPhCH2S)H152]

), A fragment ion at m/z409 corresponding to the loss ofO-galloyl, and gallic acidat m/z 169 was also identified. Which suggested that peak2 is a benzylthioether of epigallocatechin-3-O-gallate. Peak3 (Fig. 4c), eluting at 12.0 min, showed a molecular ion[MH] at m/z 493 with characteristic fragment ions[(MPhCH2S)H]

at m/z 369, and other two frag-ment ions at m/z 433 and 309, respectively. Because therewas no more structural information, the certain structure

of Peak 3 is not clear. Peak 4 (Fig. 4d), eluting at12.5 min showed [MH] atm/z563, with a characteristicintense fragment ions [(MPhCH2S)H]

at m/z 439,the intense fragment ions atm/z411 resulting from the lossof galloyl residue, and the intense fragment ions at m/z287corresponding to the loss of galloyl residue and continuedloss of a PhCH2S unit, suggesting that peak 4 was abenzylthioethers of epicatechin-3-O-gallate. The ratios ofpeak 1, peak 2, peak 3 and peak 4 were 1:7:1: 3 by the peakarea on HPLC.

3.4. Acid hydrolysis

The acid hydrolysis of the HMWT in both methanol andwater solution gave a deep red coloration, confirming thepresence of a significant amount of condensed tannin withproanthocyanidin structure. The UVvisible spectra of acidhydrolysis solution in methanol showed a strong absor-bance at 540 nm. However, the acid hydrolysis solution inwater showed absorbance at 450 nm and 540 nm in theUVvisible spectra. HPLC analysis (Fig. 5) of the acidhydrolysis products showed that there were four anthocya-nins (a, b, c, d) in methanol acid hydrolysis solution with theratios of 5:2:3:1 by the peak area on HPLC. But only two ofthem (a, c with the ratio of 3:2) were present in water acid

hydrolysis solution. HPLCESI/MS/MS analysis in posi-

Fig. 3. High performance liquid chromatogram of thiolysis products ofHMWT (detected at 280 nm).

Fig. 4. ESI (MSMS scan) spectra of thiolysis degradation products ofHMWT; (a) corresponding to the parent irons at m/z 427; (b) corre-sponding to the parent irons at m/z563; (c) corresponding to the parentirons at m/z493; (d) corresponding to the parent irons at m/z 579.


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tive ion mode further identify the four acid hydrolysis prod-

ucts as (a) delphinidin (M+ 303), (b) an unknown com-pound (M+ 319), (c) petunidin (M+ 317), and (d) cyanidin(M+ 287). With HPLCESI/MS/MS analysis in negativeion mode, gallic acid, EGCG and gallic acid derivativeswere determined to be present in methanol acid hydrolysissolution at 280 nm. A molecular ion peak at m/z 184 withcharacteristic MS2 fragment ion [(MCH3)H]

at m/z169 was identified as gallic acid methyl ester. And anunknown compound showing [MH] at m/z 317 with acharacteristic MS fragment ion [(MC8H8O3H]

at m/z164 was also determined to be present in methanol acidhydrolysis solution at 280 nm. The Chemical Structure of

thiolysis degradation and acid hydrolysis products ofHMWT were shown inFig. 6.

3.5. Hydroxyl radical scavenging activities

Among the oxygen radicals, hydroxyl radical is the mostreactive and induces severe damages to the adjacent biomol-ecules. The scavenging effect of HMWT against hydroxylradical was investigated by using two Fenton reactions.

Fig. 7 showed the hydroxyl radical scavenging effects ofHMWT by the 2-deoxyribose oxidation method, whichwas indicated as the inhibition rate. The hydroxyl radicalscavenging activity of HMWT was increased in a dose-dependent manner within the range of concentrations from0.1 to 1.5 mg/ml. The highest hydroxyl radical scavenging

activity (90.5%) were found with 1.5 mg/ml of HMWT,when compared with that of GSP (84.8%) and LMWT(20.72%) at the same concentrations respectively (P< 0.05).

Fig. 8showed the hydroxyl radical scavenging effects ofHMWT by using the salicylic acid method, the results ofwhich were also indicated as the inhibition rate. The hydro-xyl radical scavenging activity was increased from 24.9% to87.7% in a dose-dependent manner within the range of con-centrations from 0.1 to 1.1 mg/ml of HMWT. The highest(P< 0.05) hydroxyl radical scavenging activity (87.7%) wasfound with 1.1 mg/ml of HMWT, when compared withthat of GSP (76.3%) and LMWT (24.4%) at the same con-centrations respectively. The results obtained above using

two different methods indicated that the HMWT from per-simmon pulp has very strong scavenging effects againsthydroxyl radical.

3.6. Superoxide anion radical scavenging activities

The scavenging activities of HMWT against superoxideanion radical were measured using the 1,2,3-trihydroxy-

(1) R=OH, R =OH, R =H

(2) R=OH, R =H, R=O-galloyl

(3) Unknown

(4) R=H, R =H, R =O-galloyl

(1) R1=OH, R

2=OH

(2) Unknown

(3) R1=H, R

2=OCH

3

(4) R1=H, R

2=OH

a

b

Fig. 5. Chemical structure of thiolysis degradation and acid hydrolysisproducts of HMWT: (a): thiolysis degradation products, (b): acidhydrolysis products.

Fig. 6. HPLC chromatogram of methanol solution acid hydrolysisproducts (a), water solution acid hydrolysis products (b) absorbance at

540 nm is shown.

0

20

40

60

80

100

A B C D E F G H I J

Sample

Inhibiton

%

Fig. 7. Hydroxyl radical scavenging activity of HMWT, LMWT and GSPwith 2-deoxyribose oxidation system. AH were different concentration ofHMWT (0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5 mg/mL); I, 1.5 mg/mL of GSP;J, 1.5 mg/mL of LMWT. Values are means SD (n= 3).

0

20

40

60

80

100

A B C D E F G H

Sample

Inhibiton%

Fig. 8. Hydroxyl radical scavenging activity of HMWT, LMWT and GSPwith salicylic acid system. AF was different concentrations of HMWT(0.1, 0.3, 0.5, 0.7, 0.9, 1.1 mg/mL); G. 1.1 mg/mL of GSP; H. 1.1 mg/mL

of LMWT. Values are means SD (n= 3).


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benzene autoxidation system and the results were indicatedas the inhibition rate of HMWT on superoxide anion radi-cal. As shown inFig. 9, the superoxide anion radical scav-enging activity was increased in a dose-dependent mannerwithin the range of concentrations from 0.5 to 5 mg/ml ofHMWT. The scavenging activity of HMWT at 5 mg/ml

against superoxide anion radical was found to be higher,but not markedly, than that of GSP at the same concentra-tion. Furthermore, the scavenging activity of LMWT at5 mg/ml was found to be 9.82%, statistically significantlower when compared with HMWT and GSP. These resultsshowed that the HMWT have strong scavenging effectsagainst superoxide anion radicals.

3.7. Antioxidant activity in linoleic acid system

Lipid peroxidation is known to be one of the reactionsset into motion as a consequence of the formation of freeradicals in cells and tissues. Membrane lipids are abundantin unsaturated fatty acids. Linoleic acid is especially thetarget of lipid peroxidation. The antioxidant effects ofHMWT on the peroxidation of linoleic acid were investi-gated and the results were presented in Fig. 10. HMWTexhibited inhibitory activity on the peroxidation of linoleic

acid in a dose-dependent manner. With the concentrationof 0.10% in the final reaction mixture, the inhibitory rateof HMWT on linoleic acid peroxidation reached 66.93%,much higher than that of GSP (37.33%). Quite differentlyfrom HMWT and GSP, LMWT was found to acceleratelipid peroxidation as shown inFig. 10.

4. Discussion

Generally, persimmon tannin was reported to be highmolecular weight compound. The separation and structuraldetermination of persimmon tannin are very difficult due tothe strong hydrogen bond-forming ability of this molecu-lar, thus aggregating each other and firmly adsorbing tovarious absorbents and other materials in various chroma-tographies because of a surprisingly large number of phe-nolic hydroxyl groups of persimmon tannin.

In the present study, macroporous adsorption resincombined with ultrafiltration were successfully employed

to isolate and purify high molecular persimmon tanninand low molecular phenolic compounds from crudeextract; and a considerable yield of HMWT (87%) wasobtained. After ultrafiltration, a hump without anyresolved small peaks on the 280 nm chromatographic pro-file of the retentate was observed, suggesting that smallmolecular phenolic compounds could be separated effec-tively from HMWT by this simple, low cost and time-sav-ing procedure.

GPC combined with HPLCESIMS determination wasdeveloped for the analysis of high molecular weight tanninfrom persimmon. GPC could directly provide information

on molecular weight range of HMWT (Schofield, Mbugua,& Pell, 2001). Final confirmation of the monomer unit con-stitution of the structures was obtained from thiolysis deg-radation and acid hydrolysis. When condensed tannins areheated in the presence of acid and benzyl mercaptan, thechain-ending unit at the bottom is released as an unsubsti-tuted flavanol, whereas all internal units appear as benzylthioethers (Kennedy, Matthews, & Waterhouse, 2000). Inprinciple, this would permit determination of both chainlength and composition of tannin when thiolysis productsare analyzed by HPLCMS. The thiolysis condition, at60 C for 2 h, was suitably optimized after conducting atvarious temperatures. It may concluded that the condensedtannin from this Chinese persimmon consists of (epi) gallo-catechin, epigallocatechin-3-O-gallate, epicatechin-3-O-gal-late, and an unknown monomer unit, with the molecularweight of 1.28 104 Da in Mn and 1.39 10

4 Da in Mw.The result is different from that ofMatsuo and Ito (1978).

Many diseases including cancer, cataracts and cardio-vascular diseases et al. are associated with oxidative dam-ages from hydroxyl radical, superoxide anion radical andlipid peroxidation. Recently, polyphenols in plant tissuessuch as grape seeds, apple pulp, hawthorn and persimmonleaf have shown significantly strong antioxidant activities.It has been reported that persimmon seeds extracts have

a stronger radical scavenging activity than grape seeds

0

10

20

30

40

50

60

70

80

A B C E FD G H

Sample

Inhibiton%

Fig. 9. Superoxide anion radical scavenging activities of HMWT, LMWTand GSP. AF was different concentration of HMWT (0.5, 1.0, 1.5, 2.0,2.5, 5 mg/ml); G, 5 mg/mL of GSP; H, 5 mg/mL of LMWT. Values aremeans SD (n= 3).

-10

0

10

20

30

40

5060

70

80

A B C D E F

Sample

Inhibition%

Fig. 10. Inhibition effect on Linoleic acid lipid peroxidation of HMWT,LMWT and GSP. AD were different concentration of HMWT (0.02%,0.06%, 0.08%, 0.10%) in reaction system; E, 0.10% of GSP in reactionsystem; F, 0.10% of LMWT in reaction system. Values are means SD

(n= 3).


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extracts (Ahn et al., 2002). Some researches investigatedthe radical scavenging activity of methanol extracts of per-simmon folium,Laminaria japonica, andUndaria pinnatifi-da against DPPH (1,1-diphenyl-2-picrylhydrazyl) andfound the persimmon extracts was the most potent (Han& et al., 2002). However, recent investigations have shown

some differences between different test systems for thedetermination of antioxidant activity (Schlesier, Harwat,Bohm, & Bitsch, 2002). Therefore, it was recommendedto use at least two test systems to evaluate the efficacy ofantioxidation. In the present study, we used four differentsystems to investigate whether high molecular weightcondensed tannin are the main antioxidants in persimmonpulp. HMWT from persimmon showed strong antioxidantactivity in all of the test systems and was found to bemore potent antioxidant than LMWT from persimmonand oligomeric proanthocyanidins from grape seeds(GSP), the former of which was even found to acceleratelipid peroxidation. Hagerman and et al. (1998) suggested

that tannin, or polymeric polyphenolics, may be muchmore potent antioxidant than are simple monomeric phen-olics duo to the high molecular weight and the proximity ofmany aromatic rings and hydroxyl groups. The resultsobtained here were in agreement with the suggestion andfurther indicated that the high molecular weight condensedtannins were the major antioxidants in persimmon pulp.

Acknowledgments

This study was partially supported by the GuangdongAgricultural Science and technology program

(2007A020200003-2) and we are grateful to Erning Yangfor his support in HPLCMS and Xiaoman Gu for herhelp in GPC.

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