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Industrial Crops and Products 37 (2012) 520– 526

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal homep age: www.elsev ier .com/ locate / indcrop

tudies on bioactivities of tea (Camellia sinensis L.) fruit peel extracts: Antioxidantctivity and inhibitory potential against �-glucosidase and �-amylase in vitro

uefei Wang, Shuangru Huang, Shuhong Shao, Lisheng Qian, Ping Xu ∗

epartment of Tea Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China

r t i c l e i n f o

rticle history:eceived 5 May 2011eceived in revised form 24 July 2011ccepted 26 July 2011vailable online 16 August 2011

a b s t r a c t

Tea fruit peel is the main byproduct during manufacture of tea seed oil. The great increase in tea seed oilproduction in recent years brings the challenge of finding application for tea fruit peel. The aims of thisstudy were to obtain tea fruit peel extracts enriched with bioactive compounds by several solvent extrac-tion methods and to evaluate their antioxidant activity and inhibitory potential against �-glucosidase and�-amylase in vitro. Flavonoids and phenolics were accumulated in ethyl acetate, butanol, and chloroformfractions, and these fractions possessed much better antioxidant activities, including scavenging effect

eywords:ea fruit peelxtractionntioxidant activity

nhibition

on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) cation radicals (ABTS•+), and reducing activity, compared with those of the 75% ethanol extractand water fraction. Moreover, the ethyl acetate, butanol, and chloroform fractions exhibited excellentinhibitory activity against �-glucosidase, much stronger than that of the positive control (acarbose).

ed m

-Glucosidase-Amylase

These fractions also show

. Introduction

Approximately 3000 million kg of tea (Camellia sinensis (L.)untze, Theaceae) are produced and consumed each year, making it

he second most popular beverage in the world (Khan and Mukhtar,007). And as the popularity of tea has increased, the amount ofnother product of tea plant, tea fruit, has greatly increased as well.n China, over 1000 million kg of tea fruit are produced per yearTian and Shi, 2004).

Increasing amounts of tea fruit peel have been produced dur-ng the manufacture of tea seed oil, and this was recognized byhe Ministry of Health, China, as a new food resource in 2009.oth government and scientists have proposed the recycling of

ndustrial byproducts recently on both environmental grounds andor potential applications of the bioactive phytochemicals, likeavonoids and phenolics, in such byproducts. Flavonoids and phe-olics are main plant-derived biocompounds and are known asatural antioxidants due to their redox properties, allowing themo act as free radical scavenger, hydrogen donors, reducing agentsnd metal ion chelators (Javanmardi et al., 2003). Natural antiox-dants have received considerable attention due to their ability torevent human body against oxidative stress induced by imbal-

nce between generation and removal of reactive oxygen speciesnd retard the progress of many chronic diseases (Ozsoy et al.,008). Recently, natural antioxidants which can inhibit some key

∗ Corresponding author. Tel.: +86 571 88982217; fax: +86 571 88982217.E-mail address: zdxp@zju.edu.cn (P. Xu).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.07.031

ild inhibition on �-amylase activity.© 2011 Elsevier B.V. All rights reserved.

enzymes, like �-glucosidase and �-amylase, linked to postpran-dial hyperglycemia have attracted a lot of interest as a potentialapproach for curing type 2 diabetes mellitus (DM) (Hays et al., 2008;Kwon et al., 2008; Ranilla et al., 2010).

Previous studies have revealed that many industrial byproducts,like peels, skins, and hulls, from a wide range of food or medi-cal plants, were rich in flavonoids and phenolics, and the extractsenriched with such biocompounds exhibited effective antioxidantactivity and can be used as natural antioxidants in the food andpharmaceutical industry (Goli et al., 2005; Makris et al., 2007).However, to date, tea fruit peel resource has not been utilized, andmost of it is abandoned as industrial waste. Moreover, little atten-tion has been paid to the bioactive compounds of tea fruit peel andtheir bioactivities.

The objectives of this research were to establish a procedureto obtain tea fruit peel extracts enriched with bioactive com-pounds, and to evaluate the bioactivities, including antioxidantactivities and in vitro inhibitory potential against �-glucosidase and�-amylase, of the extract and solvent-extracted fractions.

2. Materials and methods

2.1. Chemicals

Folin–Ciocalteu’s phenol reagent, gallic acid, dimethyl sul-

foxide (DMSO), butylated hydroxytoluene (BHT), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tripyridyl-s-triazine(TPTZ), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′, 4′′-disulfonic

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cid sodium salt (Ferrozine), baker’s yeast �-glucosidase (EC.2.1.20), porcine pancreatic �-amylase (EC 3.2.1.1), p-nitrophenyl--d-glucopyranoside (pNPG) and all the catechin standards wereurchased from Sigma Chemical Co. (MO, USA). Methanol and ace-onitrile of HPLC grade were purchased from Tianjin Shield Co.Tianjin, China). All other chemicals were analytical grade and pur-hased from Sinopharm Chemical Reagent Co. (Shanghai, China).

.2. Raw materials

Tea fruit was collected from Panban tea garden (Zhejiang, China)n December. The fruit was washed in distilled water three timesnd then the peel was separated. The separated peel was oven-ried at 40 ◦C for 24 h. After that the dried peel was milled intoowder by a pulverizer (XB-02, Xiaobao Machinery Co., Zhejiang,hina) and passed through a 20 mesh sieve. The resulting tea fruiteel powder was stored in the refrigerator at −20 ◦C until needed.

.3. Preparation of 75% ethanol extract and fractions

Tea fruit peel powder (50 g) was refluxed with 10 vol. (v/w) of5% ethanol at 75 ◦C for 2 h, and the extraction was repeated threeimes. The extracts were filtered through filter paper, concentratedith a vacuum evaporator, and dried with a freeze drier. The freeze-ried ethanol extract (75% EtOH, 18.23 g) was dissolved in distilledater (300 ml), and fractionated with 100 ml of chloroform, ethyl

cetate, butanol, and water, respectively. The fractionated samplesere then concentrated with a vacuum evaporator and furtherried with a freeze drier. Then the freeze-dried chloroform frac-ion (C, 1.09 g), ethyl acetate fraction (E, 2.15 g), butanol fraction (B,.92 g) and water fraction (W, 5.41 g) were obtained.

.4. Determination of total flavonoid and phenolic content

The total flavonoid content was determined according to theethod of Kim et al. (2003). Samples were prepared at 20 mg/ml

n 75% ethanol. Then 0.5 ml of the sample solution was added to 10 ml volumetric flask containing 5 ml absolute ethanol. Then.3 ml of NaNO2 (5%, w/v) was added to the flask. After 5 min, 0.3 mll (NO3)3 (10%, w/v) was added. At 6 min, 4 ml of NaOH (1 M) wasdded to the mixture and adjusted to 10 ml with absolute ethanol.he mixture was thoroughly mixed and the absorbance was mea-ured at 510 nm. Catechin was used for constructing the standardurve. The total flavonoid content was calculated as catechin equiv-lents. The total phenolic content was determined according tohe Folin–Ciocalteu method modified by Ranilla et al. (2010). One

illiliter of sample solution was transferred into a 10 ml volumet-ic flask and mixed with 6 ml of distilled water. To each sample,.5 ml of Folin–Ciocalteu reagent (50%, v/v) was added and mixed.fter 5 min, 1 ml of Na2CO3 (5%, m/v) was added to the mixture anddjusted to 10 ml with distilled water. After standing for 60 min atoom temperature, the absorbance was measured at 760 nm. Gal-ic acid was used for constructing the standard curve. The totalhenolic content was expressed as gallic acid equivalents.

.5. DPPH• scavenging activity

The DPPH• scavenging activity of the tea fruit peel extracts wasetermined by the method of Mohsen and Ammar (2009), with a

light modification. One milliliter of the tested samples at variousoncentrations (125–2400 �g/ml) was added to 3 ml of ethanolicPPH• solutions (0.1 mM). The increasing of absorbance was mea-

ured at 517 nm after incubation for 30 min at 30 ◦C in the dark.

Products 37 (2012) 520– 526 521

BHT was used as the positive control. The DPPH• scavenging effectwas calculated as follows:

DPPH• scavenging effect (%) =(

1 − Asamp

Acont

)× 100

where Asamp and Acont were defined as absorbance of the sampleand the control (BHT), respectively. EC50 values (mg/ml), the effec-tive amount of the sample needed to scavenge DPPH• by 50%, weredetermined from the plotted graphs of scavenging activity againstthe concentration of the extracts.

2.6. ABTS•+ scavenging capacity

ABTS•+ assay was carried out according to the method of Caiet al. (2004). The ABTS•+ solution was prepared by mixing 7 mMABTS and 2.45 mM potassium persulfate and incubating in the darkat room temperature for 12 h. The ABTS•+ solution was then dilutedwith water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. ABTS•+

solution (3 ml) was added to 0.1 ml of the test sample with vari-ous concentrations (125–2400 �g/ml) and mixed vigorously. Theabsorbance was measured at 734 nm after standing for 6 min. BHTwas used as the positive control. The ABTS•+ scavenging effect wascalculated as follows:

ABTS+• scavenging effect (%) =(

1 − Asamp

Acont

)× 100

where Asamp and Acont were defined as absorbance of the sampleand the control (BHT), respectively. EC50 values (mg/ml), the effec-tive amount of the sample needed to scavenge ABTS•+ by 50%, weredetermined from the plotted graphs of scavenging activity againstthe concentration of the extracts.

2.7. Reducing activity

The ferric-reducing antioxidant power (FRAP) assay was per-formed according to a modified method of Benzie and Strain (1999).Briefly, the working FRAP reagent was prepared by mixing 10 vol.of 300 mM acetate buffer (pH 3.6) with 1 vol. TPTZ (10 mM) in HCl(40 mM) and with 1 vol. of FeCl3 (20 mM). Freshly prepared FRAPreagent was warmed at 37 ◦C, and a reagent blank reading wastaken at 593 nm. Subsequently, 0.6 ml of sample was added to theFRAP reagent (4.5 ml). A second reading at 593 nm was performedafter 8 min. The initial blank reading with the FRAP reagent alonewas subtracted from the final reading of the FRAP reagent withthe sample to determine the FRAP value of the sample. A standardcurve was prepared using different concentrations (25–1500 �M)of FeSO4·7H2O. BHT was used as the positive control. The reducingability of the extracts was expressed as the equivalent to that of1 �M FeSO4·7H2O.

2.8. Ferrous chelating activity

The chelating effect of the tea fruit peel extracts on ferrous ionwas assayed according to the method of Yuan et al. (2008) with aminor modification. One milliliter of sample at different concentra-tions (62.5–2400 �g/ml) was mixed with 1 ml FeSO4 (0.1 mM) for30 s, then 1 ml of ferrozine (0.25 mM) was added and the mixturewas kept for 10 min at room temperature. The absorbance of themixture was determined at 562 nm. The chelating effect on ferrousion was calculated as follows:

Chelating effect (%) =(

1 − Asamp)

× 100

Acont

where Asamp and Acont were defined as absorbance of the sampleand the control (EDTA), respectively. Then EC50 values (mg/ml),the effective amount of the sample needed to chelate ferrous ion

5 s and Products 37 (2012) 520– 526

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Fig. 1. HPLC chromatograms of catechin standards (a) and ethyl acetate fraction(b). GC, (+)-gallocatechin; EGC, (−)-epigallocatechin; C, (+)-catechin; EC, (−)-

Total flavonoids and phenolic contents of 75% ethanol extract oftea fruit peel were 39.64 and 53.12 mg/g of dry weight of extract,respectively (Fig. 2). Total flavonoid content of the chloroform, ethyl

22 Y. Wang et al. / Industrial Crop

y 50%, were determined from the plotted graphs of scavengingctivity against the concentration of the extracts.

.9. ˛-Glucosidase inhibitory activity

The �-glucosidase inhibitory activity of the tea fruit peelxtracts was determined according to the method described bypostolidis and Lee (2010) with a slight modification. A mixturef 50 �l of sample and 100 �l of 0.1 M phosphate buffer (pH 6.9)ontaining �-glucosidase solution (1 U/ml) was incubated in 96ell plates at 25 ◦C for 10 min. After preincubation, 50 �l of 5 mM

NPG solution in 0.1 M phosphate buffer (pH 6.9) was added toach well at timed intervals. The reaction mixtures were incubatedt 25 ◦C for 5 min. Before and after incubation, absorbance wasecorded at 405 nm by microplate reader (SpectraMax M5, Molec-lar Devices, CA, USA). Acarbose was used as the positive control.he �-glucosidase inhibitory activity was expressed as inhibitionercent and was calculated as follows:

nhibition (%) =(

1 − �Asamp

�Acont

)× 100

here Asamp and Acont were defined as absorbance of the samplend the control (acarbose), respectively.

.10. ˛-Amylase inhibition activity

The �-amylase inhibitory activity of the tea fruit peel extractsas determined according to a modification of the method ofanilla et al. (2010). A total of 250 �l of sample and 125 �l of 0.02 Modium phosphate buffer (pH 6.9 with 6 mM NaCl) containing �-mylase solution (0.5 mg/ml) was incubated at 25 ◦C for 10 min.fter preincubation, 250 �l of 1% starch solution in 0.02 M sodiumhosphate buffer (pH 6.9 with 6 mM NaCl) was added to each tubet timed intervals. The reaction mixtures were then incubated at5 ◦C for 10 min. The reaction was stopped with 0.5 ml of dinitros-licylic acid color reagent. The test tubes were then incubated in

boiling water bath for 5 min and cooled to room temperature.he reaction mixture was then diluted after adding 5 ml of dis-illed water, and absorbance was measured at 540 nm. Acarboseas used as the positive control. The �-amylase inhibitory activityas calculated as follows:

nhibition (%) =(

1 − �Asamp

�Acont

)× 100

here Asamp and Acont were defined as absorbance of the samplend the control (acarbose), respectively.

.11. Determination of catechin content

Tea catechins, including C, (+)-catechin; CG, (+)-catechin gal-ate; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EGC,−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; GC, (+)-allocatechin; GCG, (+)-gallocatechin gallate, were determinatedccording to the HPLC method described by Liang et al. (2007).he HPLC analysis conditions were as follows: injection volume,0 �l; column, TC-C18 5 �m, 4.6 mm × 150 mm (Agilent Technolo-ies Inc., CA, USA); oven temperature, 28 ◦C; mobile phase A,cetonitrile/acetic acid/water (6/1/193); mobile phase B, acetoni-rile/acetic acid/water (60/1/139); gradient elution, mobile phase

increased from 30% to 85% by linear gradient during the early5 min and holding at 85% for further 5 min; flow rate, 1 ml/min;

etecting wavelength, 280 nm. Catechins were identified and quan-ified by comparing their retention time and peak area with thosef the authentic standards. The HPLC chromatograms of catechintandards and sample were given in Fig. 1. The catechins contents

epicatechin; EGCG, (−)-epigallocatechin gallate; GCG, (+)-gallocatechin gallate; ECG,(−)-epicatechin gallate; and CG, (+)-catechin gallate.

in the extracts from tea fruit peel were expressed as mg/g of dryweight of extract.

2.12. Statistical analysis

All the experiments were carried out in triplicate. The resultswere expressed as means ± SD and evaluated by analysis of vari-ance (ANOVA) followed by Tukey’s studentized range test carriedout on the SAS system for windows V9, and p < 0.05 was regardedas statistically significant. Pearson’s correlation coefficients weredetermined by SPSS (version 16.0).

3. Results and discussion

3.1. Total flavonoid and phenolic content

Fig. 2. Total flavonoid and phenolic content of tea fruit peel extract and fractions.75% EtOH, 75% ethanol extract; C, chloroform fraction; E, ethyl acetate fraction; B,butanol fraction; and W, water fraction. (a−e) Means different letters in the samecolumn are significantly different at p < 0.05.

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cetate, butanol, and water fractions were 39.46, 122.83, 133.95nd 10.58 mg/g of dry weight of extract, respectively. Similarly, theotal phenolic content of the chloroform, ethyl acetate, butanol,nd water fractions were 111.47, 133.86, 176.40, and 24.46 mg/gf dry weight of extract, respectively. Total flavonoid and pheno-ic contents in the butanol fraction were the highest among theour fractions, followed by that in the ethyl acetate and chloroformractions.

These results are in good agreement with previous studies onther plant materials, like Cinnamomum osmophloeum twigs (Chuat al., 2008) and olive leaves (Lee et al., 2009). It may be thatavonoids and phenolic compounds in these materials can be easilyxtracted by ethyl acetate and butanol due to their similar polar-ty. These results also indicate that the high content of flavonoidsnd phenolic compounds in the butanol, ethyl acetate, and chloro-orm fractions may account for their stronger antioxidant activitiesompared with those of the 75% ethanol extract and water fraction.

.2. Antioxidant properties

.2.1. DPPH• scavenging activityDPPH• has been used extensively to evaluate antioxidant effects.

he scavenging ability of BHT, 75% ethanol extract, and variousractions is shown in Fig. 3a. It can be observed that the butanolnd ethyl acetate fractions exhibited much stronger scavengingbility than that of BHT at all the tested concentrations. Besides,ompared with BHT, the chloroform fraction showed a slightlyeaker scavenging ability at the low concentrations (150 �g/ml

nd 300 �g/ml), whereas the scavenging effect of chloroform frac-ion increased faster, and was much higher at concentrationsf 1200 �g/ml and 2400 �g/ml. The EC50 values of the butanol,thyl acetate and chloroform fractions were 250 �g/ml, 340 �g/mlnd 710 �g/ml, respectively, and were significantly lower than62 �g/ml of BHT (p < 0.05), while the EC50 value of BHT was much

ower than 1217 �g/ml of 75% ethanol extract. In addition, theater fraction exhibited poor scavenging of DPPH•, and showed

he weakest scavenging ability among those of all the extracts inhe tested concentration range (150–2400 �g/ml). The maximumcavenging ratio of water fraction was 27.24% at the highest con-entration of 2400 �g/ml.

Pearson’s correlation coefficients between DPPH• scavengingbility and total flavonoids was 0.914 (p < 0.05), and that betweenPPH• scavenging ability and total phenolics was 0.915 (p < 0.05),hich demonstrated that DPPH• scavenging ability may depend on

he amount of total flavonoids and total phenolics in the extracts.hese results are in agreement with other reports (Razali et al.,008; Lee et al., 2009; Ozsoy et al., 2009), in which extracts enrichedith flavonoids or phenolics showed much better DPPH• scaveng-

ng ability than those of the other extracts. These extracts can reactith free radicals to convert them to more stable products, termi-ating radical chain reactions.

.2.2. ABTS•+ scavenging activityABTS•+ is more reactive than DPPH• and unlike the reactions

ith DPPH•, which involves H atom transfer, the reactions withBTS•+ involve an electron transfer process (Kaviarasan et al.,007). In addition, the ABTS•+ model can be used to assess thecavenging activity for both polar and non-polar samples, and thepectral interference is lessened as the absorption maximum oftensed is a wavelength not normally encountered in natural productsRe et al., 1999). Therefore, it was considered necessary to furtherssess the ethanol extract and isolated fractions against the ABTS•+.

The ABTS•+ scavenging activity of 75% ethanol extract and theifferent fractions is depicted in Fig. 3b. At a concentration of00 �g/ml, the scavenging effect of butanol and ethyl acetate frac-ions, were 45.20% and 37.94%, respectively, which were much

Products 37 (2012) 520– 526 523

higher that those of the 75% ethanol extract (12.80%), the chlo-roform fraction (16.48%), and the water fraction (5.38%). At aconcentration of 2400 �g/ml, the scavenging effect of all testedsamples, except that of the water fraction, was over 95%, and BHTshowed the highest scavenging ability (99.32%), followed by thechloroform fraction (99.23%) and the ethyl acetate fraction (98.7%),but no significant difference was observed between them (p > 0.05).Among the extracts and BHT, the scavenging activity, comparingthe EC50 values, on ABTS•+ decreased in the following order: BHT(<150 �g/ml) > ethyl acetate fraction (217 �g/ml) > butanol frac-tion (258 �g/ml) > chloroform fraction (543 �g/ml) > 75% ethanolextract (849 �g/ml). However, the water fraction showed the weak-est scavenging activity on ABTS•+ at all tested concentrations, andthe maximum scavenging ratio was 46.66% at the highest con-centration of 2400 �g/ml. These results are consistent with thoseof DPPH• scavenging assay, with the antioxidant efficiency of theextracts corresponding to the content of flavonoids (Pearson’s cor-relation coefficient was 0.895, p < 0.05) and phenolics (Pearson’scorrelation coefficient was 0.883, p < 0.05) in the extracts.

3.2.3. Reducing activityFRAP measures the antioxidant effect of any substance in the

reaction medium as reducing ability. Reducing ability is consideredthe ability of a natural antioxidant to donate electrons (Shi et al.,2009). As shown in Fig. 3c, all tested samples exhibited reducingability in a concentration-dependent manner. The extracts per-formed differently in reducing TPTZ-Fe (III) complex to TPTZ-Fe(II) complex, with the butanol fraction having the strongest reduc-ing power with a value of 1437 �M FeSO4·7H2O at a concentrationof 300 �g/ml, which was not significantly different from that ofBHT at the same concentration (p > 0.05), followed by that of theethyl acetate fraction and the chloroform fraction. Meanwhile, thewater fraction showed the weakest reducing power, increasing veryslowly with increasing concentration in the range 37.5–300 �g/ml.

Many reports have revealed a direct correlation between antiox-idant activity and reducing ability of certain extracts (Duh et al.,1999), and bioactive compounds, like phenolics, are reported tohave the total antioxidant capacity of blood plasma through ferric-reducing antioxidant power assay in vitro (Serafini et al., 2003).In this assay, a significant positive correlation was found betweenFRAP values and total flavonoids (Pearson’s correlation coefficientwas 0.003, p < 0.05), and between FRAP values and total pheno-lics (Pearson’s correlation coefficient was 0.902, p < 0.05). Thus, theresults obtained from the FRAP assay also indicate that fractionsenriched with flavonoids and phenolics, acting like ferric-reducingagents, have a potential to protect humans from oxidative stressinduced by excessive ferric ion.

3.2.4. Ferrous chelating activityBy forming a stable iron (II) chelate, an extract with high

chelating power reduces free ferrous ion concentration and thusdecreases the extent of the Fenton reaction which is implicatedin many diseases (Halliwell and Gutteridge, 1984). As shown inFig. 3d, not all of the extracts were able to chelate ferrous ion aswell as EDTA. Unexpectedly, it was the water fraction, which didnot perform well in previous antioxidant assays involving DPPH•,ABTS•+, and FRAP, which exhibited the best ability for chelatingferrous among all fractions and the 75% ethanol extract. A similarphenomenon was observed in a previous study on the antioxidantactivity of extracts from C. osmophloeum twigs (Chua et al., 2008),in which the water fraction also presented much better ferrous

chelating activity than those of the other extracts. The Pearson’scorrelation coefficients between ferrous chelating activity and totalflavonoids was −0.367 (p > 0.05), and that between ferrous chelat-ing activity and total phenolics was −0.589 (p > 0.05).

524 Y. Wang et al. / Industrial Crops and Products 37 (2012) 520– 526

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in the extracts from tea fruit peel. Thus the extracts enriched withphenolic compounds, like the butanol fraction, chloroform fractionand ethyl acetate fraction, can act as �-glucosidase inhibitors andhave the potential for hyperglycemia management.

ig. 3. Antioxidant activities of tea fruit peel extract and fractions. (a) DPPH• scavengctivity. Explanation of the symbols are given already in Fig. 2. BHT, butylated hydr

It is well known that ferrous chelating ability may be involvedn antioxidant activity and affect other functions that contribute tontioxidant activity (Moure et al., 2006). Therefore, at least partly,he chelating ability of the water fraction on ferrous ion may influ-nce other activities of scavenging free radicals which protect therganism against oxidative damage. Since ferrous ions are the mostffective pro-oxidants in the food system (Yamauchi et al., 1988),he good ferrous ion chelating abilities of water fraction could beeneficial.

.3. Inhibitory potential against ˛-glucosidase and ˛-amylase

.3.1. Inhibitory activity on ˛-glucosidase�-Glucosidase, a key enzyme for carbohydrate digestion, has

een recognized as a therapeutic target for modulation of postpran-ial hyperglycemia, which is the earliest metabolic abnormality toccur in type 2 DM (Lebovitz, 1998; Krentz and Bailey, 2005). Thenhibitory effects of the extracts and acarbose on �-glucosidasere shown in Fig. 4a. �-Glucosidase was strongly inhibited by theutanol, chloroform, and ethyl acetate fractions at a concentrationf 600 �g/ml, and the inhibitory effects of butanol, chloroform, andthyl acetate fractions were much stronger than that of acarboset concentrations of 37.5 �g/ml, 150 �g/ml, and 600 �g/ml. Mean-hile, the inhibitory effect of 75% ethanol extract on �-glucosidaseas not significantly different from that of acarbose at the high

oncentration of 600 �g/ml, whereas at relatively low levels ofoncentration (37.5 �g/ml and 150 �g/ml), 75% ethanol extracthowed weaker inhibitory capacity compared with that of acar-ose.

Previous reports have shown that bioactive compounds, likeavonoids and phenolics, from a wide range of food or medicallant sources could be effective �-glucosidase inhibitors (Matsuit al., 2006; Kwon et al., 2008; Shibano et al., 2008; Kim et al.,

010). A highly positive correlation was observed between �-lucosidase inhibitory activity and the phenolic contents of thextracts (Apostolidis and Lee, 2010). In our study, a siginificantositive correlation (Pearson’s correlation coefficient was 0.941,

tivity; (b) ABTS•+ scavenging activity; (c) reducing activity; and (d) ferrous chelatingluene; EDTA, ethylenediaminetetraacetic acid.

p < 0.05) was found between �-glucosidase inhibitory effect andtotal phenolics of the extracts, and the correaltion coefficientbetween �-glucosidase inhibitory effect and total flavonoids of theextracts was 0.745, but was insignificant (p > 0.05). These resultsindicate that the inhibitory capacity against �-glucosidase dependson the content of phenolics rather than the content of flavonoids

Fig. 4. Inhibitory effects on �-glucosidase (a) and �-amylase (b) of 75% EtOH extractand its fractions from tea fruit peel. Explanation of the symbols is given already inFig. 2. (a−f) Means different letters in the same column are significantly different atp < 0.05.

Y. Wang et al. / Industrial Crops and Products 37 (2012) 520– 526 525

Table 1The catechin content in the investigated extract and fractions from tea fruit peel (mg/g of dry weight of extract).

Samples Catechins

GC EGC C EC EGCG GCG ECG CG

75% EtOH ND 0.12 ± 0.00 d 0.21 ± 0.00d 1.41 ± 0.02d 1.23 ± 0.03d 0.03 ± 0.00d 0.55 ± 0.02d NDC ND 0.64 ± 0.02b 0.34 ± 0.01d 3.47 ± 0.06c 9.33 ± 0.41c 0.90 ± 0.02c 3.75 ± 0.11c 0.13 ± 0.00b

E ND ND 4.17 ± 0.02b 26.60 ± 0.47a 34.99 ± 1.13a 1.13 ± 0.04b 11.75 ± 0.31b NDB ND 6.94 ± 0.11a 7.55 ± 0.33a 15.00 ± 0.12b 12.78 ± 0.26b 2.15 ± 0.03a 1.59 ± 0.03a NDW ND 0.48 ± 0.02c 0.86 ± 0.01c 0.29 ± 0.00e 1.08 ± 0.02e ND ND 0.18 ± 0.01a

75% EtOH, 75% ethanol extract; C, chloroform fraction; E, ethyl acetate fraction; B, butanol fraction; W, water fraction.G ; EGCg( rent a

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C, (+)-gallocatechin; EGC, (−)-epigallocatechin; C, (+)-catechin; EC, (−)-epicatechinallate; CG, (+)-catechin gallate. ND, not detected.a−e) Means different superscript letters in the same column are significantly diffe

.3.2. ˛-Amylase inhibition activityPancreatic �-amylase is a key enzyme in the digestive system

nd catalyzes the initial step in the hydrolysis of starch, which is principal source of glucose in the diet (Tarling et al., 2008). Thenhibitory effects on �-amylase of the extracts from tea fruit peelnd acarbose are shown in Fig. 4b. As observed, all the extractsxhibited lower �-amylase inhibitory activity, compared with thatf acarbose, which showed potent inhibition of �-amylase. Theighest inhibitory effects of the 75% ethanol extract, chloroform

raction, ethyl acetate fraction, butanol fraction and water frac-ion were 11.9%, 22.3%, 30.8%, 38.3% and 8.24%, respectively, at00 �g/ml which was the highest concentration tested. The Pear-on’s correlation coefficients between �-amylase inhibitory effectnd total flavonoids of the extracts was found to be 0.941 (p < 0.05),nd that between �-amylase inhibitory effect and total phenolicsf the extracts was 0.991 (p < 0.01).

The activity of �-amylase in the small intestine correlates toostprandial glucose levels, the control of which is thus an impor-ant factor in postprandial hyperglycemia, which is linked to thenset of type 2 DM. Our results are in agreement with those ofrevious studies, which have demonstrated that plant-derived phe-olic phytochemicals have lower �-amylase inhibitory activity andtronger inhibition potential against �-glucosidase (Apostolidist al., 2006; Kwon et al., 2008; Apostolidis and Lee, 2010), and suchatural enzyme inhibitors would likely offer an attractive therapeu-ic approach to the treatment of postprandial hyperglycemia, dueo lower abdominal side effects arising from excessive inhibition ofancreatic �-amylase, which results in the abnormal bacterial fer-entation of undigested carbohydrates in the colon (Kwon et al.,

006). Therefore, these results indicate that tea fruit peel extractsnriched with flavonoids and phenolics, such as the butanol, ethylcetate, and chloroform fractions, have the potential to contributeo the management of type 2 diabetes, because of their potent inhi-ition against �-glucosidase and mild inhibition of �-amylase.

.4. HPLC analysis

Generally, similar phytochemicals are present in different partsf plant, like leaves, fruits, and seeds. Catechins are the mainolyphenols in tea leaves, and have also been detected in otherarts of tea plant, like flowers (Yang et al., 2009) and seedsRavichandran, 1993). The content of catechins in the 75% ethanolxtract and its fractions from tea fruit peel are shown in Table 1.ll eight catechins, except GC, were detected in the extracts. EC

1.41 mg/g of dry weight of extract) and EGCG (1.23 mg/g of dryeight of extract) in 75% ethanol extract were found to be theajor catechins. GC (0.03 mg/g of dry weight of extract) and CG

not detected in the 75% ethanol extract) were minor catechins. EC,

GCG and ECG were mainly extracted by ethyl acetate, while EGCnd C accumulated in the butanol fraction.

The results from HPLC analysis showed the major tea cate-hins are also present in tea fruit peel, and accumulate in ethyl

G, (−)-epigallocatechin gallate; GCG, (+)-gallocatechin gallate; ECG, (−)-epicatechin

t p < 0.05.

acetate and butanol fractions, which may partly explained theoutstanding antioxidant activity and enzymes inhibitory abilityof the ethyl acetate and butanol fractions (Figs. 3 and 4). How-ever, the total amount of catechins was much lower than thoseof total flavonoids and total phenolics measured by colorimetry.This could be explained, at least partly, by the performance ofcolorimetry which tended to be less accurate than that of HPLC,but it still strongly suggests that some other flavonoids or phe-nolic compounds in addition to catechins existing in tea fruit peelwere extracted by 75% ethanol and dispersed in various fractions. Inprevious studies, gallic acid, quercetin, kaempferol, myricetin andsome other bioflavonoids have been found to present in tea leaves(Wang et al., 2000) and flowers (Yang et al., 2009). However, thereis little information about if these biocompounds exist in tea fruitpeel or not. Thus, isolation and characterization of the bioactivecompounds of tea fruit peel should be further studied.

4. Conclusions

In the present study, tea fruit peel extracts, especially ethylacetate, butanol, and chloroform fractions, not only possessedremarkable scavenging activity on DPPH• and ABTS•+, and reducingactivity, but also exhibited excellent inhibitory potential against �-glucosidase and mild inhibition of �-amylase in vitro. Moreover,flavonoids and phenolics were found to be mainly responsible forthese bioactivities of the extracts from tea fruit peel according tocorrelation determination. HPLC analysis implied that some otherflavonoids or phenolic compounds in addition to tea catechinsexisted in tea fruit peel. These results indicate that tea fruit peelcould be utilized as a renewable bioresource to develop functionalfood, health-promoting and potential antidiabetic agents. Further-more, ethanol extraction could be an effective step for obtainingnatural antioxidants from tea fruit peel although further optimiza-tion of extraction is required.

Acknowledgement

This work was supported by the Science and Technology Depart-ment of Zhejiang Province, PR China (No. 2010C32051).

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