Accepted Manuscript

Effects of tea polyphenols and different teas on pancreatic α-amylase activity in vitro

Xiaoping Yang, Fanbin Kong

PII: S0023-6438(15)30254-1
DOI: 10.1016/j.lwt.2015.10.035
Reference: YFSTL 5030

To appear in: LWT - Food Science and Technology

Received Date: 5 July 2015

Revised Date: 10 October 2015
Accepted Date: 13 October 2015

Please cite this article as: Yang, X., Kong, F., Effects of tea polyphenols and different teas on pancreatic
α-amylase activity in vitro, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.10.035.

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 ACCEPTED MANUSCRIPT

1 Effects of tea polyphenols and different teas on pancreatic α-amylase activity in vitro

2 Xiaoping Yang a, b, Fanbin Kong b, *

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3 Key Laboratory of Horticultural Plant Biology (Huazhong Agricultural University),

4 Ministry of Education, Wuhan, 430070, P.R. China

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b
5 Department of Food Science and Technology, College of Agricultural and Environmental

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6 Sciences, The University of Georgia, Athens, Georgia, 30602-2610, United States

7 * Corresponding author: E-mail: fkong@uga.edu; Tel.:+1-7065427773; Fax: +1-7065421050

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8 Abstract

9 The effects of tea polyphenols (TP) and different types of teas (green, black and oolong tea)

10 processed from the same fresh leaves on pancreatic α-amylase activity were studied using

11 potato starch and cooked potato as substrates. The results showed that, with the both

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12 substrates, low concentration of TP significantly increased α-amylase activity while high

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13 concentration inhibited it by non-competitive fashion. In addition, the extents of enzymatic

14 activation and inhibition were different when TP was pre-incubated with α-amylase or starch,

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15 respectively. The interaction of TP with enzyme/starch led to decreased antioxidant capacity.

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16 Results also showed that all the three types of teas significantly enhanced α-amylase activity
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17 for a wide range of concentrations (0.34-27.14 mg/mL), and green tea showed the highest

18 activation effect. It is concluded that high concentration of TP exhibits mild inhibitory effect
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19 against α-amylase, while green tea, black tea and oolong tea enhance α-amylase activity,
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20 which may be due to other constituents in the tea, enhancing α-amylase activity that
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21 counteracts the inhibitory effect of TP.

22 Keywords: tea polyphenols, tea, α-amylase, starch, antioxidant

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23 1. Introduction

24 Tea is a popular drink around the world, as it is the second most commonly consumed

25 beverage worldwide only behind water. Tea has many health benefits such as lowering

26 cholesterol levels, preventing cancer, and improving the immune system function (Sinija &

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27 Mishra, 2008). Studies have also shown that tea may help regulate the digestion and drinking

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28 tea benefits digestive health. In fact, tea has been used for thousands of years as a

29 postprandial digestive aid in India and China (Underwood, 2012). The beneficial effects of

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30 tea on the digestive system may be related to tea polyphenols (TP, mainly containing

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31 catechins). Studies have shown that epigallocatechin-3-gallate (EGCG), a catechin, may help
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32 regulate the digestive system as it reduces inflammation in the gastrointestinal tract and

33 colitis models (Oz, Chen, & Villiers, 2013).

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34 Despite the consensus that tea improves digestive health, the effects of tea and TP on
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35 digestive enzymes, especially α-amylase, are not yet agreed upon. For example, Hara and
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36 Honda (1990) found that TP inhibited α-amylase in a noncompetitive fashion, while other

37 researchers found that TP and green tea extract had little or no inhibition of α-amylase (Kwon,
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38 Apostolidis, & Shetty, 2008; Gao, Xu, Wang, Wang, & Hochstetter, 2013). In addition, the
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39 inhibition of α-amylase seems to be dependent on the type of tea. It has been reported that
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40 black tea had substantial inhibitory effect against α-amylase whereas green tea had low or no

41 inhibitory effect (Koh, Wong, Loo, Kasapis, & Huang, 2010; Quesille-Villalobos, Torrico, &

42 Ranilla, 2013). In another study, Wu, Ding, Xia and Tu (2010) found that TP increased the

43 activity of pancreatic α-amylase. Liu, Wang, Peng and Zhang (2011) also found that green TP

44 increased the digestion rate of corn starch. From the findings, it is difficult to draw a

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45 conclusion on how TP and teas affect α-amylase activity. It was noted that some studies

46 investigated purified TP, and others used teas extracts. Since the effects of TP and tea on

47 α-amylase activity might be different due to the existence of other components in the tea, a

48 study comparing TP and tea extract should help obtain insight information about the role of

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49 tea in modulating the enzyme activity. It was also noted that different materials and

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50 experimental conditions were involved in the existing studies, such as source of teas,

51 incubation order (i.e. the sequence of adding substrate and enzyme to TP/ tea extract.) and the

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52 concentrations of TP/tea extract tested.

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53 To compare the effect of different types of teas on the enzyme activity, it is important to
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54 have the teas processed from the same fresh tea leaves because the composition and total

55 phenolic content in different fresh tea leaves maybe markedly different, thus affecting the
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56 composition and total phenolic content in tea extracts (Lin, Tsai, Tsay, & Lin, 2003). It was
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57 noted that the teas were mostly obtained from local stores in the existing researches. It was
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58 difficult to ensure the same fresh tea leaves were used. Moreover, teas used in some studies

59 were in the form of tea bag, which could contain additives that might affect enzyme activity.
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60 Similarly, different concentrations of TP and tea extracts were used in existing studies making
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61 it difficult to compare the results. A study covering a wide scope of the concentration of TP
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62 and tea extracts should give more complete information about the impact of tea on enzyme.

63 In addition, the incubation order of TP and tea extracts might also affect α-amylase activity

64 because polyphenol can interact with both proteins and polysaccharides forming a binary or

65 ternary complex (Luck et al., 1994), but there is no study available in the literature about the

66 effect of the incubation order on α-amylase activity.

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67 The objective of this study was to systematically investigate the effect of TP and different

68 types of tea extracts on the α-amylase activity as affected by concentration of TP/tea extracts

69 and incubation orders. The study aimed to provide comprehensive information regarding the

70 impact of tea on α-amylase activity. In this study, three types of teas, including green tea,

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71 black tea and oolong tea, were processed with the same fresh tea leaves. TP and these teas

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72 were compared for their influence on the α-amylase activity, covering a wide range of

73 concentrations and the two different incubation orders. In addition, the change in the

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74 antioxidant capacity of TP in the supernatant of the enzyme hydrolysis system was also

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75 studied to understand the interactions among TP, starch and enzyme.
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76 2. Materials and Methods

77 2.1. Materials

78 Tea polyphenols (TP) from green tea (purity of 98 g/100g) was purchased from Nanjing

79 Qingze Medical Technological Development Co. Ltd (China). Porcine pancreatic α-amylase

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80 (EC 3.2.1.1), potato starch, 3, 5-dinitrosalicylic acid were purchased from Sigma Chemical

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81 Co. (St. Louis, MO). All reagents and chemicals used were of analytical grade.

82 2.2. Processing of tea

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83 Green tea, black tea and oolong tea were respectively processed at the tea factory of

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84 Huazhong Agricultural University (HZAU), Wuhan, China. Briefly, a terminal bud and two
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85 young leaves were firstly picked from Camellia sinensis bushes in tea garden at HZAU. Then,

86 with these fresh leaves, green tea was processed by fixing, rolling and drying, and black tea
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87 was processed by withering, rolling, fermenting and drying, and oolong tea was processed by
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88 sunshine withering, tedding fresh leaves, rocking green, stir-fry green, rolling and drying (Shi
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89 1997). These teas were crushed, sieved through 0.42 mm pore size sieve and stored at -20

90 for further use.

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91 2.3. Effect of TP on α-amylase activity

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92 2.3.1 Influence of TP concentration

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93 The α-amylase inhibition assay was conducted with potato starch as substrate using the

94 method of Zhao, Iyer, Flores, Donhowe and Kong (2013). TP was dissolved in phosphate

95 buffer (pH 6.9, 20 mmol/L). Potato starch solution (3.0 g/100 mL) was prepared by

96 dissolving potato starch in phosphate buffer (pH 6.9, 20 mmol/L) and gelatinized for 20

97 minutes at 80 . A series of tubes was added with 300 µL serial 2-fold dilutions of TP

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98 solution, including 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00, and 20.00 mg/mL, respectively,

99 and 300 µL of α-amylase (100 U/mL) in phosphate buffer (pH 6.9, 20 mmol/L). The tubes

100 were incubated in a water bath at 37 for 20 minutes, and added with 1mL 3.0 g/100 mL

101 potato starch solution. The reaction mixture was incubated at 37 for 20, 60, 120 minutes

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102 and then the reaction were stopped with 1.5 mL of dinitrosalicylic acid reagent. Thereafter,

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103 the mixture was boiled for 5 minutes and cooled to room temperature. Then the reaction

104 mixture was diluted by adding 6 mL of deionized water and the absorbance was measured at

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105 540 nm using a UV-vis spectrophotometer (Evolution 300, Thermo Fisher Scientific Inc,

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106 USA). The α-amylase activity was expressed as U/mL, where one unit was defined as the
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107 amount of enzyme required to release one mol glucose equivalent per minute under the above

108 assay condition. The inhibitory effect was calculated using following formula: Inhibitory
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109 effect (%) = (the α-amylase activity of control - the α-amylase activity of sample)/ the
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110 α-amylase activity of control×100.

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111 2.3.2 Influence of pre-incubation of TP and starch

112 The effect of incubation order on α-amylase activity was tested with potato starch as
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113 substrate using the same method as described above, except that 300 µL of TP solution and
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114 1mL of potato starch solution were mixed first and incubated at 37 for 20 minutes,
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115 followed by the addition of 300 µL of α-amylase solution.

116 2.4. Inhibition mode of TP on the α-amylase

117 The catalysis kinetics of α-amylase in the presence of TP at different concentrations (0, 10,

118 and 20 mg/mL) were performed using the method of Chethan, Sreerama and Malleshi (2008).

119 300 µL of TP solution and different amounts of α-amylase solution (100 U/mL, 50, 100, 150,

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120 200, 250, and 300 µL) were added to a series of tubes, followed by the addition of phosphate

121 buffer to make the final volume 600 µL. The tubes were incubated at 37 for 20 minutes,

122 and added with 1mL of potato starch solution (3.0 g/100 mL). The reaction mixture was

123 incubated at 37 for 30 minutes. Lineweaver-Burk (LB) analysis of α-amylase in the

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124 presence of TP (0, 10, and 20 mg/mL) was performed according to the methods of Chethan,

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125 Sreerama and Malleshi (2008). The inhibition was measured with increasing concentrations

126 of potato starch as a substrate (0.5, 1.0, 2.0, and 3.0 g/100 mL). 300 µL of TP solution and

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127 300 µL of α-amylase solution (100 U/mL) were mixed first and incubated at 37 for 20

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128 minutes, followed by the addition of 1mL of different concentrations of potato starch solution
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129 for 30 minutes at 37 . The type of inhibition was determined by LB plot analysis of the data,

130 which were calculated from the results according to Michaelis-Menten kinetics.
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131 2.5. Change in the antioxidant capacity of TP during enzymatic hydrolysis

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132 Similar to above, 300 µL of TP solution was incubated with 300 µL of α-amylase solution
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133 (100 U/mL) in phosphate buffer at 37 for 20 minutes. Then the mixture was added with 1

134 mL 3.0 g/100 mL potato starch solution, and incubated at 37 for 120 minutes. The
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135 ferric-reducing antioxidant capacity (FRAP) of TP in the supernatant during enzymatic

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136 hydrolysis was measured using the method of Benzie and Strain (1996), and results were
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137 expressed as mmol/L Fe2+ equivalents. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·)

138 scavenging activity was determined according to the method described by Zhu et al. (2002) ,

139 and results were expressed as ascorbic acid equivalent antioxidant capacity (AEAC). The

140 hydroxyl radical (·OH) scavenging activity was measured by the deoxyribose method

141 (Aruoma, 1994) modified by Hagerman et al. (1998), and results were expressed as

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142 scavenging percentage (%).

143 2.6. Preparation of tea extracts and determination of total phenolic contents

144 25.0 g of green tea, black tea and oolong tea were respectively soaked in 100 mL boiling

145 deionized water for 30 minutes (Zhong, 1989). The mixtures were centrifuged and the

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146 supernatants were collected and diluted to obtain 100 mL tea extracts. The tea extracts were

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147 further diluted 1.0-fold, 1.25-fold, 1.67-fold, 2.5-fold, 5-fold, and 25-fold to obtain a series of

148 tea extracts. Total phenolic contents in them were measured with Folin-Ciocalteu’s method

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149 using gallic acid for the standard curve, and results were expressed as mg of gallic acid

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150 equivalents (GAEs) per millilitre of tea extract (mg GAEs/mL).
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151 2.7. Effect of tea extracts on α-amylase activity

152 The effects of tea extracts on α-amylase activity were measured according to the method
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153 described above in section 2.3.1. 300 µL of the above tea extracts was incubated with 300 µL
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154 of α-amylase, then added with 1mL of potato starch solution. The activation effect was
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155 calculated using following formula: Activation effect (%) = (the α-amylase activity of sample

156 - the α-amylase activity of control)/ the α-amylase activity of control×100.

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157 2.8. Change of antioxidant capacity in tea extracts during enzymatic hydrolysis
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158 The change of antioxidant capacity of different tea extracts in the supernatant during
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159 enzymatic hydrolysis was measured as described in section 2.5.

160 2.9. Statistical analyses

161 All data were expressed as the mean ± SD of three replications of the experiment.

162 Statistical analysis was performed by one-way analysis of variance followed by LSD test.

163 Significant difference was considered at p < 0.05.

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164 3. Results

165 3.1. Effect of TP on α-amylase activity

166 As shown in Fig. 1, TP concentration and incubation order significantly affected α-amylase

167 activity. When pre-incubated with α-amylase prior to addition of potato starch (Fig. 1a), TP

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168 significantly increased α-amylase activity at low concentrations. The activation effect

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169 increased with the increase of TP concentration and gradually reached a peak at 2.50 mg/mL

170 of TP (0.47 mg/mL in reaction mixture), and the maximum activation magnitude of the

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171 activity was above 200%. After that, the enzyme activation magnitude gradually decreased

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172 with increasing TP concentration. TP significantly inhibited α-amylase activity in a dose-
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173 dependent manner when its concentration was more than 10.00 mg/mL (1.88 mg/mL in

174 reaction mixture). When TP was pre-incubated with starch (Fig. 1b), the initial increase in
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175 enzyme activity at low concentrations of TP was also observed but within a much lower
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176 concentration limit of 1.25 mg/mL (0.23 mg/mL in reaction mixture), and the increase in
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177 enzyme activity was also very limited (max. about 12.5%). TP started to significantly inhibit

178 α-amylase activity in a dose-dependent manner at TP concentration over 2.50 mg/mL (0.47
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179 mg/mL in reaction mixture). As an indicator of inhibitory potency, the concentrations

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180 required for 50% inhibition of enzyme activity (IC50) were calculated from the dose-response
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181 curves, and the IC50 value was 13.78 mg/mL (2.58 mg/mL in reaction mixture) when TP was

182 pre-incubated with α-amylase, and 9.83 mg/mL (1.84 mg/mL in reaction mixture) with starch,

183 respectively.

184 3.2. Inhibition kinetics of TP on α-amylase

185 Fig. 2a depicts the catalysis kinetics of α-amylase in the presence and absence of TP. It

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186 shows that the lines intersect at the origin with different slopes, and the higher the TP

187 concentration, the smaller the slope of the line is, which shows that TP is a reversible

188 inhibitor against α-amylase because it only decreases the catalysis velocity of α-amylase as

189 indicated by the change of the slope.

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190 It is well-known that an inhibitor is noncompetitive when increasing the concentration of

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191 substrate results in a family of lines with a common intercept on the 1/V axis but with

192 different slopes (Zhao, Iyer, Flores, Donhowe, & Kong, 2013). Fig. 2b depicts the effect of

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193 TP on α-amylase activity as presented in Lineweaver-Burk type plots, which suggests that the

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194 inhibition of α-amylase by TP is noncompetitive.
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195 3.3. Change in the antioxidant capacity of TP during enzymatic hydrolysis

196 The antioxidant capacity of TP in the supernatant during enzymatic hydrolysis was
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197 measured. The pre-incubation with α-amylase significantly decreased the antioxidant
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198 capacity of TP in the supernatant. However, the subsequent enzymatic hydrolysis

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199 significantly increased the antioxidant capacity in the supernatant although it was still

200 significantly lower than that of untreated TP. The antioxidant capacity of TP after 60 minutes
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201 of enzymatic hydrolysis was significantly higher than that after 20 minutes and 120 minutes
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202 of enzymatic hydrolysis. The TP content in the supernatant was also measured and a similar
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203 changing trend was observed (data not shown). A strong positive linear correlation (FRAP:

204 R2 0.996; DPPH· scavenging activity: R2 0.738; ·OH scavenging activity: R2

205 0.906) existed between the concentration and the antioxidant capacity of TP.

206 3.4. Effect of tea extracts on α-amylase activity

207 In this study, the effect of three types of teas processed from the same fresh tea leaves on

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208 α-amylase activity was studied as a comparison of that of TP. A series of concentrations of

209 the extracts of green tea, black tea and oolong tea was prepared with different total phenolic

210 contents. As expected, total phenolic contents in green tea extract was significantly higher

211 than that in oolong tea extract, and that in oolong tea was significantly higher than that in

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212 black tea (Table 1).

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213 As shown in Fig. 3a-b, all the tea extracts significantly increased α-amylase activity for all

214 the test concentrations, and the activation effect was dependent on their concentration and the

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215 type of tea. Green tea extract showed the highest activation effect, and the activation

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216 magnitude increased with the increasing extract concentration until reaching a maximum, and
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217 then gradually decreased. Oolong tea extract showed a similar trend but the activation

218 magnitude was significantly lower. The activation magnitude of black tea extract increased
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219 continually and leveled off with the increasing concentration.

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220 Like TP, the incubation order of tea extract also had significant effect on α-amylase activity.
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221 Fig. 3c shows the effect of incubation order for green tea extract with potato starch as

222 substrate. It can be seen when green tea extract was pre-incubated with starch, the maximum
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223 activation magnitude of the activity was 405%, where tea extract was diluted 1.67-fold and
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224 total phenolic content was 16.03 mg/mL (3.01 mg/mL in reaction mixture). For the case
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225 where tea extract was pre-incubated with α-amylase, the maximum activation magnitude was

226 367%, which was obtained by tea extract with 2.5-fold dilution and the phenolic content

227 10.44 mg/mL (1.96 mg/mL in reaction mixture).

228 3.5. Antioxidant capacity of different teas during enzymatic hydrolysis

229 Table 1 shows the total phenolic content and antioxidant capacity for the three tea extracts

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230 without dilution. The total phenolic contents follow the order: green tea> oolong tea> black

231 tea. The antioxidant capacities, as expressed by the FRAP and the radical scavenging activity

232 against DPPH· and ·OH, were well correlated with their total phenolic contents. The

233 antioxidant capacities of the three tea extracts and their total phenolic contents in the

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234 supernatant were also measured during enzymatic hydrolysis (data not shown). The results

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235 showed that the change of the antioxidant capacities of the three tea extracts showed a similar

236 trend to that of their total phenolic contents in the supernatant during enzymatic hydrolysis: a

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237 significant decrease when enzyme was just added followed by a gradual increase with

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238 continued enzymatic hydrolysis. Strong positive linear correlations were also observed
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239 between the antioxidant capacity and total phenolic content in the extracts of green tea

240 (FRAP: R2 0.992; DPPH· scavenging activity: R2 0.765; ·OH scavenging activity:
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241 R2 0.977 ), black tea (FRAP: R2 0.996; DPPH· scavenging activity: R2

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242 0.663; ·OH scavenging activity: R2 0.992) as well as oolong tea (FRAP: R2 0.989;
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243 DPPH· scavenging activity: R2 0.734; ·OH scavenging activity: R2 0.964).

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244 4. Discussion

245 Chinese people have consumed tea for thousands of years, holding the belief that drinking

246 tea can help digestion, and some studies have shown that tea increases digestive enzyme

247 activity and can be used as digestion aid (Tagliazucchi, Verzelloni, & Conte, 2005; Wu, Ding,

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248 Xia, & Tu, 2010). However, some recently published studies imply that drinking tea hinders

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249 digestion (He, Lv, & Yao, 2006; Forester, Gu, & Lambert, 2012) because TP is capable of

250 binding and precipitating protein through non-covalent interaction (Dreosti, 2000), and the

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251 interaction between TP and digestive enzyme can change the molecular configuration of

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252 enzyme, leading to the loss of enzyme activity and thereby reducing food digestibility. The
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253 disagreement within literature about the effect of TP and teas on enzyme activity might be

254 related to the different TP concentrations and incubation orders involved in these studies. The
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255 source of tea was also responsible for the contradictory results because most of the existing
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256 research were conducted with teas purchased from market. It is also noted that tea extracts
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257 and purified TP may possess different effect due to the existence of other components in teas.

258 In this project, we tested the effect of TP and the extracts of green tea, black tea as well as
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259 oolong tea processed with the same fresh tea leaves on α-amylase activity in vitro. A wide
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260 range of concentrations of TP and tea extracts were tested, comparing two different
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261 incubation orders and using potato starch as substrate.

262 The results from this study showed that the effects of TP and tea extracts on α-amylase

263 activity were complex, and concentration, incubation order as well as tea type all

264 significantly affected α-amylase activity. When the ratio of starch to enzyme remained

265 unchanged, TP at low concentrations (below about 8 mg/mL, 1.50 mg/mL in reaction mixture)

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266 increased α-amylase activity, whereas high concentrations (over 10 mg/mL, 1.88 mg/mL in

267 reaction mixture) inhibited enzyme activity. The IC50 value against α-amylase was 13.78

268 mg/mL (2.58 mg/mL in reaction mixture) when TP was pre-incubated with α-amylase, and

269 9.83 mg/mL (1.84 mg/mL in reaction mixture) with starch, respectively, which was similar to

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270 the report of Gao, Xu, Wang, Wang and Hochstetter (2013), who showed that the IC50 value

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271 of TP against α-amylase was 1.37 mg/mL. We also tested the effect of TP with cooked potato

272 as substrate, and found the similar effect (data not shown), and the maximum increase in

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273 enzyme activity was 84.75% for 20 minutes of enzymatic hydrolysis, which was lower than

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274 that with potato starch (134.27%) as substrate. These results suggested that TP only had a
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275 mild inhibitory effect against α-amylase at high concentrations. Hara and Honda (1990)

276 reported a strong inhibition effect of TP against α-amylase with an IC50 value ranging 0.5-120
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277 µg/mL. The disagreement may be due to different experimental conditions: a relatively low
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278 content of starch was used in their test to react with TP before enzyme was added. The starch
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279 might have been bound to TP leading to reduced hydrolysis efficiency.

280 The results also showed that the extracts of green tea, black tea and oolong tea increased
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281 the enzyme activity for all the test concentrations (Fig. 3a-b) despite of the high total phenolic
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282 contents they contained (maximum concentration was 27.14 mg/mL in green tea extract, 8.05
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283 mg/mL in black tea extract and 18.98 mg/mL in oolong tea extract). The strong enzyme

284 activation effect of tea extracts might be explained by the composition of teas. Although

285 polyphenols were the major component of tea extracts, other components, such as ions and

286 caffeine, were also present in tea extracts and might affect the activity of α-amylase. Several

287 studies have reported that ions such as Cl- (Aghajari, Feller, Gerday, & Haser, 2002), Co2+,

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288 Ca2+ (Saboury, 2002) and Ba2+ (Usha, Krishna, Muni, & Hemalatha, 2011) can enhance

289 α-amylase activity and tea extracts contain ions in abundance (Cabrera, Artacho, & Giménez,

290 2013). It was also reported that caffeine could enhance α-amylase activity (Kashani-Amin,

291 Yaghmaei, Larijani, & Ebrahim-Habibi, 2013). The content of caffeine in the tea extracts of

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292 green tea, black tea and oolong tea was tested with HPLC, which were 0.37, 0.29 and 0.32

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293 mg/mL, respectively. Therefore, we speculate that the ions and caffeine in tea extracts

294 promote the activity of α-amylase and retard the mild inhibitory effect of polyphenols against

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295 α-amylase. Fig. 3a showed that the effect of the three tea extracts on α-amylase activity was

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296 different at the same total phenolic contents, which also confirmed that the different effects
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297 were due to the other components in these tea extracts in addition to polyphenols.

298 The green tea, black tea and oolong tea were processed with the same fresh leaves and
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299 extracted in the same condition. The stronger enzyme activation effect of green tea extract
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300 than black tea extract and oolong tea extract on the same dilution (Fig. 3b) might be also
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301 related to the different components of these tea extracts for different processing technology.

302 Green tea (non-fermented tea), black tea (fully-fermented tea) and oolong tea
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303 (semi-fermented tea) are processed through attaining different levels of oxidation, and green
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304 tea possesses the highest total phenolic content and black tea possesses the highest content of
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305 polyphenol oxidation products (Jain, Manghani, Kohli, Nigam, & Rani, 2013; Yi et al., 2015).

306 It is also known that the polyphenol oxidation products are more easily to bind and

307 precipitate caffeine and metal cations than TP (Liang, Lu, & Zhang, 2002). Therefore, green

308 tea extract should contain relatively high contents of polyphenols, caffeine and metal cations,

309 and eventually demonstrate a strong net effect of activation. The activation effect of green tea

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310 extract increased at low concentration and gradually reached a peak, then gradually decreased

311 at high concentration, which is well correlated to the effect of TP (Fig. 1) which enhanced

312 enzyme activity at low concentrations and inhibited it at high concentrations. The activation

313 effect of oolong tea extract showed a similar trend to that green tea extract, but the activation

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314 magnitude was lower, which might be related to its lower content of caffeine and metal

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315 cations. Black tea extract also showed an increasing activation effect with the concentration

316 but had no decreasing phase, which is probably because the black tea extract contained much

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317 lower total phenolic content: the maximum total phenolic content was 8.05 mg/mL (1.51

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318 mg/mL in reaction mixture) for 1-fold diluted extract where TP has no obvious inhibition or
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319 activation effect on α-amylase.

320 The incubation order also significantly influenced α-amylase activity. Our study showed
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321 that when TP was pre-incubated with α-amylase prior to addition of starch, the increase in
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322 enzyme activity was significantly higher and occurred over a wider TP concentration range
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323 (up to about 8 mg/mL) than that with starch (up to 1.25 mg/mL) (Fig. 1), and the IC50 values

324 were also significantly different in the two incubation orders. A similar effect of incubation
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325 order for TP with cooked potato as substrate and for green tea extract with the both substrates
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326 was also observed (data not shown). The study revealed that TP might not only bind and
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327 precipitate α-amylase but also bind to starch, and that the binding with starch might have

328 reduced the available binding sites for the enzyme, thus affecting the α-amylase activity. This

329 was consistent with the findings of Liu et al. (2011), who reported the interaction between TP

330 and amylose, and of Xiao, Lin, Liu, Wu, Wei and Fu (2013), who also reported that the

331 highly reactive hydroxyl (OH) groups in TP interacted with the OH groups of starches

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332 leading to the formation of hydrogen bonds. Luck et al. (1994) reported that polyphenol

333 might not only form a binary complex with protein or polysaccharide, but also form a ternary

334 complex with both protein and polysaccharide, and the complexes could be reversible

335 depending on pH, temperature, and their concentrations, etc., leading to enzyme regeneration.

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336 The influence of incubation order on α-amylase activity was different for tea extracts,

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337 where the increase in enzyme activity was significantly lower when green tea extract was

338 pre-incubated with α-amylase than that with starch (Fig. 3b). The reason for the difference is

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339 not clear; but we speculate that it may also be caused by other components present in tea

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340 extract. This difference may suggest that drinking tea after a meal is more beneficial for the
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341 starch digestion than before a meal, which is consistent with Chinese belief of that drinking

342 tea after a meal helps digestion.

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343 The antioxidant capacity of TP in the supernatant was significantly decreased right after
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344 enzyme was added, then increased when enzymatic hydrolysis was continued, and the change
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345 of TP content in the supernatant had a similar trend to that of the antioxidant capacity (data

346 not shown). The initial decrease was attributed to the binding reaction of TP to enzyme, and
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347 the subsequent increase might be a result of enzyme regeneration (Luck et al., 1994). It was
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348 noted that the antioxidant capacity after 120 minutes of enzymatic hydrolysis was lower than
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349 that after 60 minutes, which might be due to that the enzymatic hydrolysis for a long time has

350 caused the degradation of TP that decreased the antioxidant capacity. Similar phenomenon

351 was observed by Chen et al. (2013) who reported decreased antioxidant capacity of tea juice

352 during in vitro digestion. The change in the antioxidant capacity of tea extracts during

353 enzymatic hydrolysis also showed a similar trend to that of TP, and strong positive linear

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354 correlations between the antioxidant capacity and total phenolic contents of tea extracts were

355 also observed, which confirmed the reaction had occurred among polyphenols, enzyme

356 and/or starch in tea-added system, indicating that the activation effect was due to other

357 components present in tea extract.

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358 5. Conclusions

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359 This study showed that TP has mild inhibitory effect against α-amylase at high

360 concentration and both TP concentration and incubation order had significant effects on

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361 α-amylase activity. Green tea, black tea and oolong tea all increased α-amylase activity and

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362 green tea has the highest activation effect. The activation effect of tea extracts on α-amylase
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363 activity may be related to other constituents such as ions and caffeine present in teas that

364 counteract the mild inhibition effect of TP. The interaction between TP and enzyme/starch
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365 also led to changed antioxidant capacity in the supernatant during enzymatic hydrolysis. To
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366 our knowledge, this is the first study comparing the effect of TP and different teas processed
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367 from the same fresh tea leaves on α-amylase activity and confirming the activation effect of

368 teas on α-amylase activity.

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369 Acknowledgments

370 This work was financially supported by the Fundamental Research Funds for the Central

371 Universities, China (grant no. 2013PY091) and the Natural Science Foundation of Hubei

372 Province, China (grant no. 2011CDB136).

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460 Table 1 Total phenolic content and antioxidant capacity of TP and tea extracts a

TP (20 mg/mL) Green tea Black tea Oolong tea

Total phenolic content 18.44±0.05b 27.14±0.94a 8.05±0.71c 18.98±0.88b

(mg GAEs/mL)

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FRAP (mmol/L Fe2+) 8.23±0.49c 16.83±0.65a 6.29±0.37d 13.58±0.58b

activity 21.37±1.25b 41.06±1.37a 21.28±1.97b 40.40±1.20a

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DPPH· scavenging

(µg/mL AEAC)

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·OH scavenging activity (%) 87.71±1.62a 86.49±1.28a 75.71±1.71c 80.67±1.92b

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461 25.0 g of green tea, black tea and oolong tea was respectively extracted with 100 mL boiling
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462 deionized water for 30 minutes, centrifuged and diluted to obtain 100 mL tea extract. Total

463 phenolic contents in the tea extracts were measured with Folin-Ciocalteu’s method, and
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464 results were expressed as mg of gallic acid equivalents per millilitre of tea extract (mg
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465 GAEs/mL). 20.00 mg/mL of TP was also expressed as mg GAEs/mL with Folin-Ciocalteu’s
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466 method. Total antioxidant capacities of TP and these tea extracts were measured. The FRAP

467 was expressed as mmol/L Fe2+ equivalents. The DPPH· scavenging activity was expressed as
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468 ascorbic acid equivalent antioxidant capacity (AEAC). The ·OH scavenging activity was
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469 expressed as scavenging percentage (%). Different letters in the same row indicate significant
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470 differences at P < 0.05.

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471 Fig.1. Effect of TP on α-amylase activity. a) 300 µL of TP was pre-incubated with 300 µL

472 of α-amylase solution (100 U/mL) at 37 for 20 minutes, followed by the addition of 1mL

473 3.0 g/100 mL starch solution at 37 for 20, 60, 120 minutes; b) 300 µL of TP was

474 pre-incubated with 1mL 3.0 g/100 mL starch solution at 37 for 20 minutes, followed by the

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475 addition of 300 µL of α-amylase solution (100 U/mL) at 37 for 20 ( ), 60 (■), 120 (▲)

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476 minutes.

477 Fig.2. The inhibition mode of TP on α-amylase. a) the catalysis kinetics of α-amylase in the

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478 presence and absence of TP [0 ( ), 10 ( ), 20 ( ) mg/mL]. 300 µL of TP solution and

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479 different amounts of α-amylase solution (100 U/mL, 50, 100, 150, 200, 250, and 300 µL)
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480 were mixed first and incubated at 37 for 20 minutes, followed by the addition of 1mL of

481 3.0 g/100 mL potato starch solution at 37 for 30 minutes. b) Lineweaver-Burk analysis of
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482 α-amylase in the presence and absence of TP. 300 µL of TP solution and 300 µL of α-amylase
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483 solution (100 U/mL) were mixed first and incubated at 37 for 20 minutes, followed by the
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484 addition of 1mL of different concentrations of potato starch solution (0.5, 1.0, 2.0, and 3.0

485 g/100 mL) at 37 for 30 minutes.

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486 Fig.3. Effect of tea extracts (a: total phenolic content; b: dilution times) and
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487 pre-incubation order (c) of green tea extract on α-amylase activity. 25.0 g of green tea
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488 (▲), black tea ( ) and oolong tea (○) was respectively extracted with 100 mL boiling

489 deionized water for 30 minutes, centrifuged and diluted to obtain 100 mL tea extract. The tea

490 extract was further diluted 1-fold, 1.25-fold, 1.67-fold, 2.5-fold, 5-fold, and 25-fold to obtain

491 a series of tea extracts. ( ) Tea extract was pre-incubated with α-amylase (▲) Tea extract

492 was pre-incubated with starch.

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493
494 a

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495

496 b

497 Fig.1

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498

499
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500

501 b

502 Fig.2

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505 a
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507 b

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509 c

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Highlights

• TP had mild inhibitory effect against α-amylase at high concentrations.

• Enzyme activity was different when TP was pre-incubated with α-amylase or

starch respectively.

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• Green tea, black tea and oolong tea all improved α-amylase activity.

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• Drinking tea could help starch digestion.

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