Inhibitory Effect of Four Types of Tea on the in vitro Digestion of Starch
Alyssa Gutierrez – University of Alabama Jiannan Feng – University of Alabama Libo Tan – University of Alabama Lingyan Kong – University of Alabama
Deposited 09/01/2020
Citation of published version:
Gutierrez, A., Feng, J., Tan, L., Kong, L. (2020): Inhibitory Effect of Four Types of Tea on the in vitro Digestion of Starch. Food Frontiers. DOI: https://doi.org/10.1002/fft2.39
© 2020 The Authors. Food Frontiers published by John Wiley & Sons Australia, Ltd and Nanchang University, Northwest University, Jiangsu University, Zhejiang University, Fujian Agriculture and Forestry University For published full text, send request to [email protected]
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2 Inhibitory Effect of Four Types of Tea on the in vitro Digestion of Starch
3
4 Alyssa Gutierrez1, Jiannan Feng2, Libo Tan2,*, Lingyan Kong2,*
5 6 1 Department of Biological Sciences, the University of Alabama, Tuscaloosa, AL 35487
7 2 Department of Human Nutrition and Hospitality Management, the University of Alabama,
8 Tuscaloosa, AL 35487
9 10 11 12 13 14 15 16 * Corresponding authors.
17 Address:
18 407 Russell Hall, 504 University Blvd, Tuscaloosa, AL 35487, USA (L. Tan)
19 482 Russell Hall, 504 University Blvd, Tuscaloosa, AL 35487, USA (L. Kong)
20 E-mail address:
21 [email protected] (L. Tan)
22 [email protected] (L. Kong)
23
24
1 25 Abstract
26 Phenolic compounds have been shown to decrease the rate of starch hydrolysis by inhibiting
27 digestive enzymes for starch. Tea, rich in phenolic compounds, has been widely reported for its
28 beneficial health effects. This study explored the inhibitory abilities of four types of tea, i.e.,
29 green tea (GT), oolong tea (OT), black tea (BT), and white tea (WT), on the in vitro enzymatic
30 digestion of potato starch (PS) and high-amylose maize starch (HAMS), respectively, using two
31 in vitro digestion methods. All teas significantly inhibited the digestion of both starches, with a
32 few exceptions at some time points. The resistant starch content was increased by all four teas in
33 HAMS, but only by BT and WT in PS. The total phenolic contents in the teas could not predict
34 the inhibititotry ability of the teas, possibly due to the structural diversity of phenolic compounds
35 and the presence of other compounds that could also alter enzymatic activity. Our results
36 suggested that tea, regardless of its type, can slow down the enzymatic digestion of starch and
37 therefore imply a potential benefit for controlling postprandial hyperglycemia.
38 Keywords
39 Starch; in vitro digestion; resistant starch; tea; total phenolic content
40
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41 1. Introduction
42 Starch is a polysaccharide comprising of a large number of glucose units linked by α-
43 glycosidic bonds. Abundant in staple foods, e.g., wheat, maize (corn), rice, and potatoes, starch
44 is the most common dietary carbohydrate and serves as a major source of energy in human diets.
45 In the gastrointestinal tract (GIT), starch is digested by a series of enzymes into glucose for
46 absorption. The kinetics of starch digestion and that of glucose release/absorption as the energy
47 source for the body determine the nutritional quality of starch and starch-containing foods
48 (Zhang & Hamaker, 2009). Rapid starch digestion may cause postprandial hyperglycemia and
49 hyperinsulinemia cycle, which is involved in a variety of metabolic diseases including metabolic
50 syndrome, obesity, type 2 diabetes, and cardiovascular diseases (Byrnes et al., 1995; Ludwig,
51 2002). Starch in certain foods, especially heavily processed foods (e.g., refined flour), can be
52 rapidly digested and results in the rapid elevation of blood glucose levels. On the contrary,
53 certain starch portions are slowly digested and even indigestible, and therefore cause a slower
54 and steadier rise in blood glucose concentration.
55 While the structure, processing, and storage conditions of starch greatly dictates its rate of
56 digestion (Jayawardena et al., 2017; Tamura et al., 2019), another critical factor is the activity of
57 starch digestive enzymes in the GIT. ⍺-Amylase is the major enzyme responsible for starch
58 digestion in the intraluminal phase. It is present in the mouth as salivary ⍺-amylase and in the
59 small intestine as pancreatic ⍺-amylase. Recent studies have demonstrated the ability of phenolic
60 compounds, a large group of plant secondary metabolites, to inhibit the activity of ⍺-amylase and
61 thus slow the rate of starch digestion (Sales et al., 2012; Xiao et al., 2013). A natural and
62 abundant source of phenolic compounds is tea, which is also known to confer other health
63 benefits such as antioxidant, anti-inflammatory, and anti-tumor properties (Pan et al., 2013; Su et
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64 al., 2016; Yen-Chen et al., 2018). Yet, previous studies that examined the effect of tea
65 polyphenols on starch digestion showed inconsistent results. For instance, Sun et al. (2018)
66 showed that tea polyphenols inhibited pancreatic ⍺-amylase activity, and Kwon et al. (2008)
67 reported that black tea (BT), green tea (GT), and oolong tea (OT) had a mild inhibitory effect
68 (ranging from 35 to 40 percentage inhibition) on pancreatic ⍺-amylase. On the contrary, Yang
69 and Kong (2016) found the same teas to enhance the enzymatic activity. This same study,
70 however, also found that high concentrations of isolated tea polyphenols mildly inhibited ⍺-
71 amylase activity. Such findings indicate that while polyphenols inhibit ⍺-amylase activity,
72 another component of tea may instead enhance it.
73 Despite the invaluable foundation set by previous studies, discrepancies clearly exist
74 regarding the effect of tea and tea polyphenols on ⍺-amylase activity and starch digestion.
75 Therefore, this study further investigated the topic via a multifaceted approach to assess the
76 effects of different types of tea, including GT, BT, OT, and white tea (WT), on the digestion of
77 two types of starch, i.e., potato starch (PS) and high-amylose maize starch (HAMS). Two
78 simulated in vitro digestion methods that haven been widely adopted by different researchers
79 were utilized. One measured the total reducing sugar contents at 1 h and 2 h upon digestion using
80 a method outlined by Flores et al. (2014), while the other measured solely the glucose contents at
81 20 min and 120 min using a method described by Englyst et al. (1992). The former method aims
82 to obtain the percent inhibition by the inhibitors, i.e., the teas. The latter method established the
83 starch digestibility profiles, where the glucose content at 20 min represents rapidly digestible
84 starch (RDS), that at 120 min is slowly digestible starch (SDS), and the remaining starch is
85 regarded as resistant starch (RS) (Englyst et al., 1992). The total phenolic content in the teas
86 were determined to evaluate a potential correlation with their inhibitory effects. The two
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87 starches, with varied in their amylose contents (approximately 25% and 80% (w/w) in PS and
88 HAMS, respectively), were utilized to assess the potential outcomes of differing amylose content
89 in starch.
90 2. Materials and Methods
91 2.1 Materials
92 High amylose maize starch (HAMS; Gelose 80) was obtained from Ingredion (Bridgewater,
93 NJ, USA). Potato starch (PS, S2004), amyloglucosidase from Aspergillus niger (EC 3.2.1.3,
94 A7095, 300 U/mL), porcine pancreatin (EC 232-468-9, P7545, 8 x USP), invertase from baker’s
95 yeast (EC 3.2.1.26, I4504, 300 U/mg), guar gum (G4129), sodium acetate trihydrate (S7670),
96 maltose (M5885), and 3,5-dinitrosalicylic acid (128848), urea (U5378), lipase (L3126, EC
97 3.1.1.3), and porcine bile extract (B8631) were purchased from Sigma-Aldrich, Inc. (St. Louis,
98 MO, USA). Potassium sodium tartrate tetrahydrate (AAA10163-30), sodium phosphate
99 monobasic (97061-942), sodium phosphate dibasic (97061-588), sodium chloride (97061-904),
100 sodium bicarbonate (BDH9280) were purchased from VWR chemicals, LLC (VWR chemicals,
101 Solon, OH, USA). English breakfast black tea (BT) and green tea (GT) (Twinings@) were
102 purchased from a local grocery store. White tea (WT) and oolong tea (OT) were purchased from
103 grocery stores in Anxi and Anji (Fujian, China), respectively. D-glucose assay kit (GOPOD
104 format, K-GLUC) was purchased from Megazyme (Wicklow, Ireland).
105 2.2 Brewing of tea samples
106 Four types of tea leaves (1.0 g) were brewed with 40.0 mL of boiling deionized (DI) water
107 for 20 min, cooled to the room temperature (20 °C), filtered with Whiteman No. 4 filter paper,
108 and labeled as GT, BT, OT, and WT, respectively.
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109 2.3 Determination of total phenolic content
110 The fast blue salts BB assay was adopted to determine the total phenolic content in the
111 brewed tea samples according to Lester et al. (2012). In detail, 1.0 mL of 100-fold diluted tea
112 samples was mixed with 1.0 mL of 0.1% (w/v) Fast blue BB for 30 s, followed by adding 0.1 mL
113 of 5.0% (w/v) NaOH. The mixture was stirred with a vortex mixer and incubated for 90 min
114 under light at room temperature (20 °C). After that, absorbance was measured at 420 nm using a
115 Mettler Toledo UV5Bio UV-vis spectrophotometer (Mettler Toledo, Columbus, OH, USA).
116 Gallic acid standard solutions were used to establish the standard curve and results were
117 expressed as milligram of gallic acid equivalents (GAE) per liter of tea.
118 2.4 In vitro digestion – Flores method
119 In the first in vitro digestion study, two types of raw starch, PS and HAMS, were subject to
120 in vitro digestion with and without the presence of brewed teas according to the method outlined
121 by Flores et al. (2014). Specifically, 1.0 mL of each tea sample was added with 0.5 g of starch
122 sample in a 50 mL test tube, followed by adding 6.0 mL of simulated intestinal fluid and 3.0 mL
123 of bile fluid. The compositions of simulated digestive juices were shown in Table 1. The pH of
124 the simulated digestive juices was adjusted with 1 M HCl or 1 M NaOH. As a control, 1.0 mL of
125 DI water was added instead of brewed tea. In order to account for the interference in absorbance
126 measurements by particles other than sugar (e.g., digestive fluid components and tea residuals), a
127 blank that contained neither starch nor tea was used. The digestion trials were conducted in an
128 orbital shaking water bath at 37 °C with a shaking speed of 160 rpm for 2 h. At 1 h and 2h of
129 digestion, 0.5 mL aliquots were collected and immediately placed in a boiling water bath for 10
130 min to deactivate the enzymes and halt the reaction.
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131 Table 1. Compositions of simulated digestive juices. Simulated intestinal juice Bile juice 500 mL DI water 500 mL DI water 7.012 g NaCl 5.259 g NaCl 0.564 g KCl 0.376 g KCl 3.388 g NaHCO3 5.785 g NaHCO3 80.0 mg KH2PO4 0.25 g Urea 50.0 mg MgCl2 15.0 g Bile salts 0.1 g Urea 9.0 g Pancreatin 1.5 g Lipase pH 8.1 ± 0.2 6.9 ± 0.1 132 After the reaction was terminated, each sample was centrifuged at 2700 g for 3 min and the
133 supernatant was then diluted to a proper concentration. The reducing sugar content was then
134 determined using the 3, 5-dinitrosalicylic (DNS) colorimetric assay. For the assay, 0.2 mL of the
135 diluted samples was combined with 0.1 mL of DNS reagent. This solution was heated in a
136 boiling water bath for 8 min and allowed to cool to room temperature (20 °C). DI water (0.9 mL)
137 was then added to each sample for further dilution. Absorbance was measured at 540 nm using a
138 Mettler Toledo UV5Bio UV-vis spectrophotometer. Absorbance readings were subtracted by the
139 reading of the blank sample to eliminate the interference of foreign particles on light scattering.
140 The standard curve was established using maltose standard solutions and the results were
141 reported as maltose equivalent (mM).
142 2.5. In vitro digestion – Englyst method
143 The second in vitro starch digestion study was administered using the method reported by
144 Englyst et al. (1992) with a few modifications. Raw starch (80 mg) alone or raw starch mixed
145 with 1 mL of brewed tea were added into 20 mL round-bottom test tubes. A test tube with no
146 starch and inhibitors was used as the blank. Also mixed into the test tubes were 10 mg guar gum,
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147 6 mm glass beads, and 4 mL of 0.1 M acetate buffer. The reaction mixtures were placed in a
148 water bath at 37 °C. Immediately before the digestion reaction, an enzyme solution, containing
149 pancreatin (46.8 mg/mL prior to centrifugation), amyloglucosidase (13 U/mL), and invertase
150 (187.5 U/mL), was prepared by dissolving or diluting the enzymes in deionized water. Enzyme
151 solution (1 mL) was added into each tube at a certain time interval and this time interval was
152 accurately followed for sampling purpose. The test tubes were capped and placed into a shaking
153 water bath at 37 °C and 150 strokes per min. After incubating for 20 and 120 min, aliquots of
154 200 µL were taken from the reaction mixture and the reaction was stopped by mixing into 900
155 µL of 66% (v/v) ethanol vigorously. These digesta samples were kept at 4 °C overnight and then
156 centrifuged (2700 g, 3 min). The glucose concentrations in the supernatant were measured using
157 the glucose oxidase-peroxidase (GOPOD) method and used to calculate the amount of digested
158 starch. The starch contents digested during the first 20 min and between 20 and120 min were
159 regarded as the RDS and SDS proportions, respectively. The amount of RS was determined as
160 the amount of starch remaining after 120 min of digestion. Results were adjusted according to
161 the measured total starch content (81.10 ± 5.72% and 100 ± 2.80% in PS and HAMS,
162 respectively), and expressed as a percentage of total starch.
163 2.6 Statistical Analysis
164 All experiments were conducted in duplicates. Data were analyzed by one-way analysis of
165 variance (one-way ANOVA) followed by Tukey multiple comparison test using the GraphPad
166 Prism software (San Diego, CA). The letters a, b, and c indicate statistically significant
167 differences, p < 0.05 (a > b > c).
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168 3. Results and Discussion
169 3.1 Effect of teas on starch digestion
170 In order to evaluate the inhibitory effect of the four different teas on in vitro enzymatic
171 starch digestion, two types of starch, i.e., PS and HAMS, which vary in their amylose content,
172 were subject to simulated in vitro digestion with and without the presence of teas. Two simulated
173 in vitro digestion methods were used to evaluate starch digestion. In the first method, the extent
174 of starch enzymatic digestion after 1 and 2 h was measured by the amount of digestion product,
175 primarily reducing sugars, released from digestion and expressed as maltose equivalent (Figure
176 1). Samples without tea (NI) served as a control that demonstrated baseline starch digestion. All
177 four teas, when compared to the control, displayed significantly (p <0.05) lower values of
178 reducing sugar from the digestion of both starch types. An exception was noted for OT in PS
179 digestion at 2 h. These results suggested that tea has an inhibitory effect on the digestion of
180 starch by ⍺-amylase in the pancreatin.
181 The digestion of HAMS and PS proceeded to approximately the same extent by 2 h, but the
182 digestion at 1 h was significantly lower for PS. The difference in the digestion behavior of
183 HAMS and PS may be attributed to the regularity and compactness of molecular arrangement in
184 the two types of starch. Zhang et al. suggested that enzymatic resistance of native granular starch
185 was probably due to the densely packed molecular order in the surface region of starch granules
186 (Zhang et al., 2015). This difference in digestion may therefore be explained by the higher
187 amylopectin content of PS. Compared with amylose, amylopectin molecules are much bulkier,
188 highly branched and more densely packed in molecular arrangement. These properties make
189 amylopectin more difficult to hydrate and be accessed by enzymes in the aqueous phase. Hence,
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190 the presence of more amylopectin in PS could have delayed the binding of ⍺-amylase and its
191 substrates, and thus lower the enzymatic digestion rate in PS.
192 193 Figure 1. Extent of HAMS and PS digested, without inhibitors (NI) and with the presence of 194 green tea (GT), black tea (BT), oolong tea (OT), and white tea (WT), expressed as maltose 195 equivalent, with and without the presence of teas after 1 h and 2 h of digestion. Significant 196 differences among treatments within each figure are denoted by different letters (a > b > c, p < 197 0.05). 198 The percent inhibition values of the teas on starch digestion at 1 and 2 h were obtained and
199 compared in Figure 2. It shows that the inhibitory ability on α-amylolysis showed no significant
200 difference among the four teas, except for a lesser inhibition on HAMS digestion by OT at 2 h.
201 Furthermore, the inhibitory percentage ranged from 8.87% to 20.45% at 2 h of digestion. The
202 lower limit indicates a particularly mild inhibitory effect.
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203 204 Figure 2. Percent inhibition of teas on HAMS and PS digestion after 1 h and 2 h of digestion 205 using green tea (GT), black tea (BT), oolong tea (OT), and white tea (WT). Significant 206 differences among treatments within each figure are denoted by different letters (a > b, p < 0.05). 207 Starch digestion is executed primarily by a series of enzymes in the GIT, including ⍺-
208 amylase and maltase (e.g., ⍺-glucosidase). The first simulated in vitro digestion method only
209 involved the former enzyme. It is important to note, however, that the latter enzyme has been
210 reported to be potently inhibited by polyphenols. In fact, studies that utilized polyphenols found
211 in oregano (Gutiérrez-Grijalva et al., 2019) and olive oil (Figueiredo-González et al., 2019)
212 demonstrated significantly greater inhibition on the activity of ⍺-glucosidase than on that of ⍺-
213 amylase. Another study reported that green tea polyphenol extracts were a stronger inhibitor of
214 ⍺-glucosidase than acarbose, which is a known ⍺-glucosidase inhibitor and common medication
215 prescribed to diabetics (Yan et al., 2018). Hence, the second simulated in vitro digestion study
216 was conducted to include the latter enzyme and obtain starch digestibility profiles as affected by
217 different teas.
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218 In the second digestive method, glucose contents of samples at 20 min and 120 min were
219 measured in order to determine starch digestibility profiles, i.e., the amounts of RDS, SDS, and
220 RS (Figure 3). Again, starches with no inhibitor (NI) were used as a control to demonstrate
221 baseline starch digestion. The RS content in HAMS was 63%, which was very close to other
222 reports in high amylose maize starches, e.g., the Hi-Maize 260 starch (Ingredion, 2019) and our
223 recent report (Gutierrez et al., 2020). The RS content in PS was notably low, yet the RDS content
224 was close to our previous reported value (Gutierrez et al., 2020). Both HAMS and PS are
225 regarded as the type 2 RS, or RS2. The resistance mechanism of RS2 was proposed to be due to
226 the densely packed molecular arrangement on the starch granule surface (Zhang et al., 2015). In
227 particular, no other RS except for high amylose starches have been classified as dietary fiber by
228 the United States Food and Drug Administration (Food and Drug Administration, 2018). Similar
229 to the results of the first digestion method, all four teas inhibited the digestion of HAMS, which
230 is expressed as a significantly increased proportion of RS and decreased RDS. The nominal RS
231 content of HAMS was increased to up to 88% by adding BT. Comparable to results of the first
232 digestion method, PS again had exceptions in exhibiting inhibitory effects, where GT and OT
233 had no appreciable effect on RDS and RS contents. When the percent inhibition was calculated
234 from these digestion data (Figure 4), the inhibitory effect of the teas was very different between
235 the two types of starches. In digesting HAMS, the extent of inhibition was much higher than that
236 observed in the first digestion method, but similarly, OT exerted the lowest inhibition upon
237 digestion for 2h. The protocols, including the enzymes and their concentrations, were different
238 between the two methods and could lead to different results. Indeed, upon digesting PS for 2 h,
239 although it agreed with the first method that there was no difference in the percent inhibition
240 values among the teas, the percent inhibition range was slightly lower than that in the first
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241 method, and GT showed the relatively low inhibition. Overall, these results were consistent in
242 that the inhibitory effect of tea is greater in the presence of starch containing higher proportions
243 of amylose. Meanwhile, the inhibitory effect on starches with lower amylose contents depends
244 on the type of tea.
245 246 Figure 3. Starch digestibility profiles, presented as rapidly digestible starch (RDS), slowly 247 digestible starch (SDS), and resistant starch (RS) contents, of raw high amylose maize starch 248 (HAMS) and raw potato starch (PS) without inhibitors (NI) and with the presence of green tea 249 (GT), black tea (BT), oolong tea (OT), and white tea (WT). Error bars show standard deviation; 250 n=2. Significant differences among treatments within each starch digestion fraction are denoted 251 by different letters (a > b > c, p < 0.05). 252
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253 254 Figure 4. Percent inhibition of teas on HAMS and PS digestion after 20 min and 2 h of digestion 255 using green tea (GT), black tea (BT), oolong tea (OT), and white tea (WT). Significant 256 differences among treatments within each figure are denoted by different letters (a > b, p < 0.05).
257 3.2 Phenolic content and inhibitory ability
258 Measurements for the total phenolic content of the teas demonstrate significant difference (p
259 < 0.05), except for between GT and OT (Figure 5). GT and OT contained the highest total
260 phenolic content, while WT had the lowest. These results are similar to that of Annunziata et al.,
261 who measured the total phenolic contents of WT, GT, and BT using the Folin-Ciocalteu method
262 (Annunziata et al., 2018). They found that WT had the lowest and GT had the highest phenolic
263 contents. The classification of tea into GT, BT, OT, and WT is based on the degree of
264 fermentation or oxidation. GT is unfermented or un-oxidized, WT is only slightly fermented, OT
265 tea is a semi-fermented tea, while BT is a fully fermented or oxidized tea (Weerawatanakorn et
266 al., 2015). Fermentation is known to reduce the phenolic content in tea (Jayasekera et al., 2014),
267 yet there are many other factors determining the phenolic content in different tea samples. In the
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268 present study, the total phenolic content was not a predictive variable for the inhibitory ability on
269 α-amylolysis, as there was no significant difference in the activity among the four teas (Figure
270 2). The inhibitory ability of the teas did not increase with their total phenolic content. One
271 possible reason is the diversity of phenolic compounds and their variance in inhibitory ability.
272 Phenolic compounds are a large group of compounds derived from a phenol, and even within the
273 same sub-group of phenolic compounds, their inhibitory effect may vary. For instance, the
274 inhibitory effect of tannins (Mkandawire et al., 2013) and proanthocyanins (Zhang et al., 2016)
275 both increased with molecular weight and degree of polymerization. Another study that
276 examined two major polyphenols of tea, i.e., (-)-epigallocatechin (EGC) and
277 (-)‑epigallocatechin‑3‑gallate (EGCG), found that the starch digestion rate was unaffected by
278 EGC but was significantly inhibited by EGCG (Xie et al., 2019). Despite the highly related
279 structures of EGC and EGCG, there is an evident disparity in their inhibitory abilities. The total
280 phenolic content determined cannot reveal the structural diversity of all phenolic compounds in
281 the teas. This could be regarded as a limitation of the present study. Quantification of individual
282 components of tea phenolics could help unravel which phenolic components may have a more
283 profound inhibitory effect on starch digestion. Secondly, the digestive processes may have
284 caused the varying results in inhibitory ability. Chen et al. reported that intestinal digestion was
285 able to degrade proanthocyandins from Acacia mearnsii bark (Chen et al., 2018). Subsequently,
286 its ⍺-glucosidase inhibitory activities were reduced by approximately 47% compared to the bark
287 extract after only gastric digestion. The various types of tea polyphenols may be degraded to
288 different extents, and their digestive products likely vary in their bioactivities. Interestingly, the
289 degradation of phenolic compounds can be beneficial. Annunziata et al. found that even though
290 WT had the lowest total phenolic content compared to GT and BT, the intestinal digestion of WT
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291 extract led to the highest duodenal and colonic bioaccessibility (Annunziata et al., 2018).
292 Enhanced bioaccessibility may yield more potent antioxidant effects. Thirdly, the lack of
293 correlation may also be attributed to other components in tea such as caffeine and ions. Caffeine
294 was reported to enhance ⍺-amylase activity (Kashani-Amin et al., 2013). Ions have been shown
295 to generate varying effects on ⍺-amylase activity. For instance, Aghajari et al. (2002) reported
296 chloride ions to enhance ⍺-amylase activity, while nitrate ions had no significant effect (Aghajari
297 et al., 2002). In relation to this theme, the content of chloride and nitrate ions in GT, BT, and WT
298 have been found to differ significantly (Mincă et al., 2013). Accordingly, it is reasonable to
299 speculate that the mild inhibitory effect in many cases was a net result of the inhibitory effect of
300 tea polyphenols and the augmenting effects of other components such as caffeine and ions.
301 Given that the composition of the other components may differ among the teas, their
302 compounding effects could also explain the lack of correlation between total phenolic content
303 and inhibition percentage.
304 305 Figure 5. Total phenolic content, expressed as mg of GAE per liter of tea, in green tea (GT), 306 black tea (BT), oolong tea (OT), and white tea (WT). Significant differences among samples are 307 denoted by different letters (a > b > c, p < 0.05).
308 4. Conclusion
309 In this study, the inhibitory effect of four types of teas, specifically GT, BT, OT, and WT, on
310 the enzymatic digestion of PS and HAMS was evaluated using two simulated in vitro digestion
16
311 methods. All teas, regardless of type, were able to significantly decrease the extent of starch
312 digestion, with a few exceptions at some time points. In the first digestion method, the extent of
313 digestion in both starches was the same at 2 h, while the slower digestion of PS at 1 h may be a
314 result of its high amylopectin content. The second method further involved an important starch
315 digestive enzyme, ⍺-glucosidase, on which polyphenolic compounds might exhibit high
316 inhibitory activities. Regarding starch digestibility profiles, RS was increased by all four teas in
317 HAMS but only by BT and WT in PS. Since HAMS displayed consistent and significant
318 decreases in starch digestion by all four teas, but PS did not, this is evidence that the amylose-
319 amylopectin contents and their molecular arrangement might contribute to the extent of
320 inhibition on digestion. Accordingly, the beneficial inhibitory effects may be most applicable in
321 the digestion of high-amylose starches. The total phenolic contents in the teas were significantly
322 different, except for green and oolong teas, but could not predict the inhibitory ability of the teas.
323 It was possibly due to the structural diversity of phenolic compounds and the presence of other
324 compounds, such as caffeine and ions, which could also alter enzymatic activity. Our in vitro
325 method utilized porcine amylase and amyloglucosidase from Aspergillus niger, and therefore the
326 teas may exhibit different results when using human enzymes or in vivo methods. Nevertheless,
327 the net results of including tea in in vitro starch digestion appear to exhibit potential as a means
328 of delaying starch digestion and glucose absorption. In addition to many known health benefits
329 of tea polyphenols, the results of this study would promote tea consumption as a potential way of
330 modulating glycemic response.
331 Conflict of Interest
332 The authors confirm that they have no conflict of interest to declare for this publication.
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333 References
334 Aghajari, N., Feller, G., Gerday, C., & Haser, R. (2002). Structural basis of α‐amylase activation 335 by chloride. Protein Science, 11(6), 1435-1441. 336 337 Annunziata, G., Maisto, M., Schisano, C., Ciampaglia, R., Daliu, P., Narciso, V., Tenore, G. C., 338 & Novellino, E. (2018). Colon bioaccessibility and antioxidant activity of white, green 339 and black tea polyphenols extract after in vitro simulated gastrointestinal digestion. 340 Nutrients, 10(11), 1711. 341 342 Byrnes, S. E., Miller, J. C. B., & Denyer, G. S. (1995). Amylopectin starch promotes the 343 development of insulin resistance in rats. The Journal of nutrition, 125, 1430. 344 345 Chen, X., Xiong, J., He, L., Zhang, Y., Li, X., Zhang, L., & Wang, F. (2018). Effects of in vitro 346 digestion on the content and biological activity of polyphenols from Acacia mearnsii 347 bark. Molecules, 23(7), 1804. 348 349 Englyst, H. N., Kingman, S., & Cummings, J. (1992). Classification and measurement of 350 nutritionally important starch fractions. European journal of clinical nutrition, 46, S33- 351 50. 352 353 Figueiredo-González, M., Reboredo-Rodríguez, P., González-Barreiro, C., Carrasco-Pancorbo, 354 A., Cancho-Grande, B., & Simal-Gándara, J. (2019). The involvement of phenolic-rich 355 extracts from Galician autochthonous extra-virgin olive oils against the α-glucosidase and 356 α-amylase inhibition. Food Research International, 116, 447-454. 357 358 Flores, F. P., Singh, R. K., Kerr, W. L., Pegg, R. B., & Kong, F. (2014). Total phenolics content 359 and antioxidant capacities of microencapsulated blueberry anthocyanins during in vitro 360 digestion. Food Chemistry, 153, 272-278. 361 362 Food and Drug Administration. (2018). The Declaration of Certain Isolated or Synthetic Non- 363 Digestible Carbohydrates as Dietary Fiber on Nutrition and Supplement Facts Labels: 364 Guidance for Industry. Retrieved 10/23/2019 from https://www.fda.gov/regulatory- 365 information/search-fda-guidance-documents/guidance-industry-declaration-certain- 366 isolated-or-synthetic-non-digestible-carbohydrates-dietary 367 368 Gutiérrez-Grijalva, E. P., Antunes-Ricardo, M., Acosta-Estrada, B. A., Gutiérrez-Uribe, J. A., & 369 Heredia, J. B. (2019). Cellular antioxidant activity and in vitro inhibition of α-
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370 glucosidase, α-amylase and pancreatic lipase of oregano polyphenols under simulated 371 gastrointestinal digestion. Food Research International, 116, 676-686. 372 373 Gutierrez, A. S. A., Guo, J., Feng, J., Tan, L., & Kong, L. (2020). Inhibition of starch digestion 374 by gallic acid and alkyl gallates. Food Hydrocolloids, 102, 105603. 375 https://doi.org/https://doi.org/10.1016/j.foodhyd.2019.105603 376 377 Ingredion. (2019). HI-MAIZE® 260 resistant starch factsheet. Retrieved 10/23/2019 from 378 https://www.ingredion.com.ar/content/dam/ingredion/pdf- 379 downloads/emea/38A%20-%20Hi-maize%20Health%20Claims%20Overview.pdf 380 381 Jayasekera, S., Kaur, L., Molan, A.-L., Garg, M. L., & Moughan, P. J. (2014, 2014/06/01/). 382 Effects of season and plantation on phenolic content of unfermented and fermented Sri 383 Lankan tea. Food Chemistry, 152, 546-551. 384 https://doi.org/https://doi.org/10.1016/j.foodchem.2013.12.005 385 386 Jayawardena, N., Herath, P. N., Watawana, M. I., & Waisundara, V. Y. (2017). Effects of 387 Processing and Storage Conditions on the in vitro Digestibility and other Functional 388 Properties of Six South Asian Starches. Journal of Food Processing and Preservation, 389 41(4), e13017. https://doi.org/10.1111/jfpp.13017 390 391 Kashani-Amin, E., Yaghmaei, P., Larijani, B., & Ebrahim-Habibi, A. (2013). Xanthine 392 derivatives as activators of alpha-amylase: Hypothesis on a link with the hyperglycemia 393 induced by caffeine. Obesity Research & Clinical Practice, 7(6), e487-e493. 394 395 Kwon, Y. I., Apostolidis, E., & Shetty, K. (2008). Inhibitory potential of wine and tea against α‐ 396 amylase and α‐glucosidase for management of hyperglycemia linked to type 2 diabetes. 397 Journal of Food Biochemistry, 32(1), 15-31. 398 399 Lester, G. E., Lewers, K. S., Medina, M. B., & Saftner, R. A. (2012). Comparative analysis of 400 strawberry total phenolics via Fast Blue BB vs. Folin–Ciocalteu: Assay interference by 401 ascorbic acid. Journal of Food Composition Analysis, 27(1), 102-107. 402 403 Ludwig, D. S. (2002). The glycemic index: physiological mechanisms relating to obesity, 404 diabetes, and cardiovascular disease. Jama, 287, 2414-2423. 405 406 Mincă, I., Josceanu, A., Isopescu, R., & Guran, C. (2013). Determination of ionic species in tea 407 infusions by ion chromatography. UPB Scientific Bulletin, Series B: Chemistry and 408 Materials Science, 75(3), 65-78.
19
409 410 Mkandawire, N. L., Kaufman, R. C., Bean, S. R., Weller, C. L., Jackson, D. S., & Rose, D. J. 411 (2013). Effects of sorghum (Sorghum bicolor (L.) Moench) tannins on α-amylase activity 412 and in vitro digestibility of starch in raw and processed flours. Journal of agricultural 413 and food chemistry, 61(18), 4448-4454. 414 415 Pan, M.-H., Lai, C.-S., Wang, H., Lo, C.-Y., Ho, C.-T., & Li, S. (2013, 2013/03/01/). Black tea 416 in chemo-prevention of cancer and other human diseases. Food Science and Human 417 Wellness, 2(1), 12-21. https://doi.org/https://doi.org/10.1016/j.fshw.2013.03.004 418 419 Sales, P. M., Souza, P. M., Simeoni, L. A., Magalhães, P. O., & Silveira, D. (2012). α-Amylase 420 inhibitors: a review of raw material and isolated compounds from plant source. Journal of 421 Pharmacy & Pharmaceutical Sciences, 15, 141-183. 422 423 Su, J., Wang, X., Song, W., Bai, X., & Li, C. (2016, 2016/12/01/). Reducing oxidative stress and 424 hepatoprotective effect of water extracts from Pu-erh tea on rats with high-fat diet. Food 425 Science and Human Wellness, 5(4), 199-206. 426 https://doi.org/https://doi.org/10.1016/j.fshw.2016.09.002 427 428 Sun, L., Gidley, M. J., & Warren, F. J. (2018). Tea polyphenols enhance binding of porcine 429 pancreatic α-amylase with starch granules but reduce catalytic activity. Food Chemistry, 430 258, 164-173. 431 432 Tamura, M., Singh, J., Kaur, L., & Ogawa, Y. (2019). Effect of post-cooking storage on texture 433 and in vitro starch digestion of Japonica rice. Journal of Food Process Engineering, 434 42(2), e12985. https://doi.org/10.1111/jfpe.12985 435 436 Weerawatanakorn, M., Hung, W.-L., Pan, M.-H., Li, S., Li, D., Wan, X., & Ho, C.-T. (2015, 437 2015/12/01/). Chemistry and health beneficial effects of oolong tea and theasinensins. 438 Food Science and Human Wellness, 4(4), 133-146. 439 https://doi.org/https://doi.org/10.1016/j.fshw.2015.10.002 440 441 Xiao, J., Ni, X., Kai, G., & Chen, X. (2013). A review on structure–activity relationship of 442 dietary polyphenols inhibiting α-amylase. Critical reviews in food science and nutrition, 443 53, 497-506. 444 445 Xie, F., Huang, Q., Fang, F., Chen, S., Wang, Z., Wang, K., Fu, X., & Zhang, B. (2019). Effects 446 of tea polyphenols and gluten addition on in vitro wheat starch digestion properties.
20
447 International Journal of Biological Macromolecules, 126, 525-530. 448 https://doi.org/https://doi.org/10.1016/j.ijbiomac.2018.12.224 449 450 Yan, S., Shao, H., Zhou, Z., Wang, Q., Zhao, L., & Yang, X. (2018). Non-extractable 451 polyphenols of green tea and their antioxidant, anti-α-glucosidase capacity, and release 452 during in vitro digestion. Journal of Functional Foods, 42, 129-136. 453 https://doi.org/https://doi.org/10.1016/j.jff.2018.01.006 454 455 Yang, X., & Kong, F. (2016). Effects of tea polyphenols and different teas on pancreatic α- 456 amylase activity in vitro. LWT-Food Science and Technology, 66, 232-238. 457 458 Yen-Chen, T., Tsung-Hai, H., Meei-Ju, Y., Wei-Lun, H., Chi-Tang, H., & Min-Hsiung, P. (2018, 459 2018/06/01/). The effects of the extract of oolong tea and its metabolites from Andraca 460 theae in high fat diet induced obese Wistar rat. Food Science and Human Wellness, 7(2), 461 120-124. https://doi.org/https://doi.org/10.1016/j.fshw.2018.05.001 462 463 Zhang, B., Dhital, S., & Gidley, M. J. (2015). Densely packed matrices as rate determining 464 features in starch hydrolysis. Trends in Food Science & Technology, 43(1), 18-31. 465 https://doi.org/https://doi.org/10.1016/j.tifs.2015.01.004 466 467 Zhang, G., & Hamaker, B. R. (2009). Slowly digestible starch: concept, mechanism, and 468 proposed extended glycemic index. Critical reviews in food science and nutrition, 49, 469 852-867. 470 471 Zhang, Y., Wong, A. I. C., Wu, J. e., Karim, N. B. A., & Huang, D. (2016). Lepisanthes alata 472 (Malay cherry) leaves are potent inhibitors of starch hydrolases due to proanthocyanidins 473 with high degree of polymerization. Journal of Functional Foods, 25, 568-578. 474 475
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