Journal of Applied Botany and Food Quality 92, 57 - 63 (2019), DOI:10.5073/JABFQ.2019.092.008 1College of Food Science and Engineering, South China University of Technology, Guangzhou, China 2Aijia Biotechnology Co. Ltd, Changsha, Hunan, China In vitro assessment of anti-diabetic potential of four kinds of dark tea (Camellia sinensis L.) protein hydrolysates Keying Su1, Xinliang Mao1, Liping Ai2, Xuewu Zhang1* (Submitted: August 23, 2018; Accepted: January 30, 2019) * Corresponding author Summary The contributions of four kinds of dark tea (Camellia sinensis L.) proteins and their hydrolysates to hypoglycemic activity were inves- tigated in vitro. Four kinds of water-extracted dark tea proteins were hydrolyzed with trypsin and Alcalase, respectively. The complete proteins had α-amylase inhibitory activity with half maximal inhi- bitory concentration (IC50) values ranging from 1.27 to 2.78 mg/mL. Most of the dark tea proteins and hydrolysates significantly inhibited α-glucosidase and dipeptidyl peptidase (DPP-IV), with IC50 values in the range of 0.0103 -1.3114 mg/mL and 0.1000 -1.3364 mg/mL, re- spectively. In general, Heimaojian (HMJ) and Qianliang (QL) hydro- lysates displayed high α-glucosidase inhibitory activity, while HMJ, Fuzhuan (FZ), and Heizhuan (HZ) hydrolysates exhibited a strong ability to inhibit DPP-IV. This study demonstrates the potential of dark tea proteins and their hydrolysates as a source of functional food and medicine for the control of type 2 diabetes. Key words: Dark tea; hydrolysis; DPP-IV; α-glucosidase; α-amylase Introduction Type 2 diabetes is one of the leading public health problems in modern society. This chronic disease increasingly exists in the aging population, especially in developed countries. By 2030, it will affect 438 million people, with 70% of cases occurring in low- to middle- income families (Yu et al., 2012). People who suffer from type 2 diabetes are strongly predisposed to atherosclerotic cardiovascular disease (CVD) (HarnedY and FitzGerald, 2013), which is a major cause of morbidity and mortality. Some synthetic medicines are used to control type 2 diabetes. One therapeutic approach is to suppress the absorption of glucose by inhibiting carbohydrate-hydrolyzing enzymes such as α-amylase, which acts on long-chain carbohydrates, and α-glucosidase, which catalyzes the cleavage of glucose from disaccharides (lebovitz et al., 1997). Acarbose and voglibose are widely known as inhibitors of α-amylase and α-glucosidase. Another mechanism is the inhibition of dipeptidyl peptidase-IV (DPP-IV) activity. DPP-IV rapidly meta- bolizes glucagon-like peptide 1 (GLP-1) and glucose-dependent in- sulinotropic polypeptide (GIP), two insulinotropic incretin hormones that enhance glucose-dependent insulin secretion and regulate post- prandial blood glucose levels (Green et al., 2004). Many DPP-IV inhibitors are used, including vildagliptin, saxagliptin, and sitagliptin. Tea (Camellia sinensis L.) is regarded as one of the most popular beverage plants consumed worldwide. It has been reported that 78% of the total amount of tea produced and consumed around the world is dark tea (full-fermented), 20% is green tea (non-fermented), and less than 2% is yellow or oolong tea (semi-fermented) (Xiao et al., 2011; li et al., 2012). One of the most important dark tea-production areas in China is Anhua County in Hunan Province, which is famous for Fuzhuan brick tea, Qianliang tea, and Heimaojian tea. Tea has many pharmacological and therapeutic properties, inclu- ding anti-diabetic effects. Stote and baer (2008) indicated that tea consumption may affect glucose metabolism and insulin signaling by enhancing insulin sensitivity and endothelial function. Many studies have verified that tea extracts significantly inhibit the activity of both α-amylase and α-glucosidase (Miao et al., 2015; PenG et al., 2015; KoH et al., 2010), enzymes that play key roles in carbohydrate diges- tion and have been recognized as therapeutic targets for modulating postprandial hyperglycemia. Gao et al. (2012) demonstrated that tea polyphenols can exert anti-oxidative and hypolipidemic effects in rats with streptozotocin-induced diabetes. HuanG et al. (2013) stated that 95% ethanol precipitate from an aqueous extract of pu-erh tea exhi- bited a remarkable inhibitory effect against α-glucosidase in vitro, as well as a significant (p < 0.05) effect on postprandial hyperglycemia in diabetic mice. Recently, CHen and Guo (2017) investigated the effects of polysaccharides and polyphenolic fractions of Zijuan tea on α-glucosidase activity and blood glucose levels. The results indicated that the polysaccharide or theaflavin fractions inhibited α-glucosidase at a greater rate than acarbose (positive control). zHou et al. (2018) reported that tea polyphenols can alter autophagy levels to improve glucose and lipid metabolism in diabetic rats with cardiomyopathy. However, the anti-diabetic effects of dark tea proteins have not yet been investigated. This study focused on four kinds of representative dark teas from Hunan Province: Heimaojian (HMJ), Fuzhuan tea (FZ), Heizhuan tea (HZ), and Qianliang tea (QL). The objective of this work was to examine the inhibitory effects of their protein extracts and hydro- lysates on the activity of α-amylase, α-glucosidase, and DPP-IV. Materials and methods Materials Dried dark tea leaves for all four kinds of tea were provided by Aijia Biotechnology Co. (Hunan Province, China). α-glucosidase from Saccharomyces cerevisiae (≥10 U/mg protein), α-amylase from Bacillus licheniformis liquid (CAS: A4862), DPP-IV Inhibitor Screening Kit (MAK203-1KT), 4-nitrophenyl α-D-glucopyranoside (pNPG) (CAS: 3767-28-0), and soluble starch were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bradford Protein As- say Kit (P0006) was purchased from Beyotime (Haimen, China). Acarbose hydrate was purchased from Aladdin. The Alcalase en- zyme (with a claimed activity of ≥200 U/mg) was purchased from Aoboxing Bio-tech Co. (Beijing, China), and trypsin (250 U/mg) was purchased from HuaQiSheng Bio-tech Co. (Guangzhou, China). 2,4,6-Trinitrobenzenesulfonic acid (TNBS, 5% in H2O) reagent was purchased from Ark Pharm (USA). Sodium dodecyl sulphate was purchased from Biofroxx. All other reagents were of analytical grade. Preparation and purification of dark tea protein extracts Dark tea leaves were milled, sieved (20 mesh), accurately weighed (25.0 g), and extracted in an ultrapure water bath (500 mL) for 30 min 58 K. Su, X. Mao, L. Ai, X. Zhang at 95 ℃. After filtration with gauze and quantitative filter papers, the filtrates were concentrated by rotary vaporization under reduced pressure at 50 ℃ to obtain the syrup extracts. Small amounts of water were then used to dilute the syrup extracts. Finally, the extracts were lyophilized, and the residues (test samples) were collected for subsequent analysis. To purify the dark tea protein, portions of the test samples were dissolved in ultrapure water at a concentration of 5 mg/mL and placed in well-prepared dialysis membranes (500 Da). Ultrapure water (250 times the volume of the samples) at room temperature was placed on the opposite side of the membrane and replaced three times (every 12 hours). After dialysis, 5% (w/v) activated carbon was added at 45 ℃ for 30 min. After filtration with quantitative filter papers, saturated ammonia sulfate solution was added (Vammonia sulfate:Vsample = 4:1), and the protein was allowed to precipitate at 4 ℃ for 12 hours. The subsidence was obtained by centrifugation at 6000 × g for 20 min at 4 ℃, and then redissolved in ultrapure water before dialysis at room temperature for 24 hours to eliminate micromolecules. Enzymatic hydrolysis of dark tea proteins Dark tea proteins were enzymatically treated with trypsin and Alca- lase following the method described previously (HarnedY and FitzGerald, 2013; WanG and zHanG, 2017), with some modifica- tions. Briefly, solutions of the crude proteins were preheated to 50 ℃ and adjusted to pH 8.0 with 2.0 M NaOH. Either Alcalase or trypsin was added at an enzyme/substrate (E/S) ratio of 1.5% (w/w). The reaction mixtures were kept in a water bath with shaker at 50 ℃ for six hours, and 2.0 M NaOH was used to maintain the pH value as monitored by a pH meter (FE30, Mettler Toledo, Switzerland). The enzymes were inactivated by heating at 90 ℃ for 20 min. Hydroly- sate samples were subsequently freeze-dried. Determination of proteins and degree of hydrolysis Proteins were determined using a Bradford Protein Assay Kit (P0006). Briefly, 5-μL test extract solutions (2 mg/mL) were mixed with 250 μL Coomassie Brilliant Blue (G-250) dye in a 96-well plate. Absorbance at 595 nm was measured within 2 hours using a micro- plate reader (Sunrise v1.05, Tecan, Switzerland). To obtain a standard curve, BSA protein standard solutions of varying concentrations (0 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1.0 mg/mL, and 1.5 mg/mL) were used. Degree of hydrolysis (DH) was monitored using the TNBS method described by SPellMan et al. (2003), with some modifications. Du- ring the course of the test, 0.5 M NaOH was used to keep the reaction at pH 7.0 as monitored by a pH meter. 0.125 mL of the test samples (2 mg/mL) were added to test tubes and mixed with 1.0 mL of sodium phosphate buffer (0.2125 M, pH 8.2). TNBS reagent (1 mL, 0.1% w/v) was then added to each tube, followed by incubation at 50 ℃ for 60 min in a water bath (protected from light). Furthermore, the reaction was stopped by adding 2.0 mL HCl (0.1 mol/L). Samples were then allowed to cool to room temperature, and absorbance values were measured at 340 nm using a microplate reader (Sunrise v1.05, Tecan, Switzerland). L-Leucine (0-2 mM) was used to generate a standard curve. DH values were calculated using the following formula: where AN1 is the amino nitrogen content of the protein substrate before hydrolysis (mg/g protein), AN2 is the amino nitrogen content of the protein substrate after hydrolysis (mg/g protein), and Npb is the nitrogen content of the peptide bonds in the protein substrate (mg/g protein). Reversed-phase high-performance liquid chromatography (RP- HPLC) analysis The peptide compositions of dark tea protein hydrolysates were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) using an Agilent 1260 HPLC system with a UV detector. Briefly, samples were diluted to 1 mg/mL in ultrapure water, filtered through a 0.22-μm syringe filter, and then injected into an analytical C18 column with a length of 250 mm, inner diameter of 4.6 mm, and particle size of 5 μm (C18-120-5 4E, Shodex, Japan). Samples were eluted at a total flow rate of 0.5 mL/min at 30 ℃, with both water (solvent A) and acetonitrile (solvent B) as follows: 15% B at 0-2 min; 15%-20% B at 2-10 min; 20%-25% B at 10-20 min; 25%-80% B at 20-30 min. Elution was monitored at 215 nm with an ultraviolet- visible (UV-vis) detector. α-Amylase inhibition assay The α-amylase inhibitory assay was performed according to the method previously described by Yu et al. (2012), with slight modi- fications. A 1% starch solution was preheated at 95 ℃ for 5 min. 10 μL of α-amylase solution (1 U/mL in ultrapure water) was pre- mixed with 20 μL of test sample solutions at different concentrations (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL) in ultrapure water. Following incubation at 37 ℃ for 15 min, 500 μL of 1% starch solution (in 0.2 M sodium phosphate buffer, pH 6.9) was added to start the reaction. The reaction was car- ried out at 37 ℃ for 5 min and terminated by adding 600 μL of DNS reagent (1% 3,5-dinitrosalicylic acid, 12% Na-K tartrate in 0.4 mol/L Na2CO3). The reaction mixture was placed in a boiling water bath for 15 min. After the samples cooled down to room temperature, absor- bance at 540 nm was determined by a microplate reader (Yu et al., 2011a; Yu et al., 2011b). Acarbose was used as a positive control. Sodium phosphate buffer (pH 6.9) was used in place of the test sam- ples for the blank. For all tests, the inhibition assay was performed in quadruplicate. The inhibition of enzyme activity was calculated as follows: Inhibition (%) = [1 − (Asample /Ablank)] × 100 α-Glucosidase inhibition assay The α-glucosidase inhibitory activity was assayed according to a pre- vious study by lin et al. (2015), with modifications and optimization. 20 μL of test extract solutions at different concentrations (0.01 mg/ mL, 0.02 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL) and of acarbose (positive control, 0.125 mg/ mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL) were pre- mixed with 10 μL of α-glucosidase (0.2 U/mL) at 37 ℃ for 20 min. Next, 40 μL of pNPG (10 mM, in 0.2 M phosphate buffer, pH 6.8) and 50 μL of 0.2 M phosphate buffer were added and incubated at 37 ℃ for 30 min. The reaction was stopped by adding 100 μL of 0.2 M sodium carbonate. Phosphate buffer was used in place of the test samples for the blank. To determine sample background interference, each sample was added to the phosphate buffer during reactions with- out the enzyme solution. Each inhibition assay was performed in qua- druplicate. Absorbance at 405 nm was measured using a microplate reader. The α-glucosidase inhibition (%) was calculated as follows: Inhibition (%) = 1 − [(Asample − Abackground)/Ablank] × 100 DPP-IV inhibition assay The DPP-IV inhibitory activity assay was performed using a DPP- IV Inhibitor Screening Kit (MAK203). The effectiveness of the test inhibitors was compared with a known DPP-IV inhibitor, sitagliptin. Before use, reagents were allowed to come to room temperature and slightly centrifuged to maintain integrity. Assays were performed as AN2 - AN1 DH% = 100 Npb Anti-diabetic potential of dark tea 59 specified by the product technical bulletin. After all reagents were diluted to the required concentrations, 50 μL of enzyme solution and 25 μL of test sample solution were premixed and incubated at 37 ℃ for 10 min; then 25 μL of DPP-IV substrate was added. After waiting 15-30 min to allow all reagents to mix, Fluorescence (FLU, λex = 360 nm, λem = 460 nm) was measured once per minute at 37 ℃ using a Cytation 5 imaging reader (BioTek, USA). A control assay was also prepared by using DPP-IV assay buffer in place of the inhi- bitor samples. To remove background interference, buffer was added instead of enzyme solution. The relative DPP-IV inhibition (%) was calculated as follows: Relative inhibition (%) =1− [(Slopesample – Slopebackground)/Slopecontrol] × 100% where Slope = (FLU2 – FLU1)/(T2 – T1) = FLU/minute Statistical analysis Results are presented as the mean of triplicate determinations ± SDs. IBM SPSS 22.0 (SPSS, Inc., Chicago, IL, USA) was used to identify significant differences (p < 0.05) between data. Results and discussion Extraction and purification of dark tea proteins The method of preparing dark tea proteins under ordinary pressure is not fully described in the existing scientific literature, but plant- derived proteins are usually extracted by one of three types of sol- vents: aqueous, alkaline, or organic (HarnedY and FitzGerald, 2013; lin et al., 2015; SioW et al., 2016). In consideration of people’s drinking habits and environmental protection concerns, aqueous sol- vent was used in this study. The concentration of protein in tea leaves is around 0.20 g/g dry matter. In this study, the yield ranged from 7.39% ± 0.87% (for the complete QL protein) to 20.3% ± 1.19% (for the HMJ hydrolysate treated with Alcalase). The presence of fiber and phenolic compounds, as well as protein glycosylation, all have inhibitory effects on protein hydrolysis, which cause misleading results in enzyme inhibitory assays. Phenolic and flavone compounds also inhibit the activity of many enzymes. Puri- fication of proteins was therefore a critical step in removing some of the inhibitory agents and increasing the accuracy of the study. Af- ter the purification treatment, the concentration of protein increased dramatically (shown in Fig. 1). The trypsin-generated QL fraction realized the largest increase at 404.8% (from 0.1080 g/g extract to 0.5452 g/g extract), followed by the complete QL protein at 321.65% (from 0.0739 g/g extract to 0.3116 g/g extract) and the complete HMJ protein at 247.16% (from 0.1767 g/g extract to 0.6134 g/g extract). The average increase in protein content for 12 protein samples was 195.11%. Enzymatic hydrolysis of protein fractions The primary sequence of the protein substrate and the specificity of the enzyme(s) used determined the types of bioactive peptides gene- rated from a particular protein. Trypsin and Alcalase have been wide- ly used on plant proteins to produce bioactive peptides (HarnedY and FitzGerald, 2013; KalYanKar et al., 2013). In this study, the TNBA assay was used to quantify the degree of hydrolysis (DH) of four kinds of dark tea protein hydrolysates. Fig. 2 illustrates that the DH of HMJ, FZ, and QL were higher when trypsin was used com- pared to Alcalase, with the exception that HZ reached a higher DH when treated with Alcalase. According to researchers, Alcalase is a proteolytic preparation derived from Bacillus licheniformis and con- sists mainly of subtilisin endoproteinase, with a minor glutamyl en- dopeptidase activity (KalYanKar et al., 2013). Subtilisin acts as a relatively nonspecific proteinase but preferentially cleaves peptide bonds after large non-β-branched hydrophobic residues (Gron et al., 1992), while trypsin specifically targets diaminocaproic acid and arginine. This may indicate that lower concentrations of diaminoca- proic acid and arginine exist in HZ. Moreover, when hydrolyzed by trypsin at a pH of 8.0 for 6 hours at 50 ℃, FZ reached the highest DH at 49% ± 0.52%, followed by HMJ (41.39% ± 1.02%), QL (35.96% ± 0.145%), and HZ (34.53% ± 0.52%). These values were higher than those obtained in previous studies; for instance, Spirulina platensis hydrolyzed by trypsin had a DH of 18.26%-20.2% (Fan et al., 2018; WanG and zHanG, 2016). The DH for proteins treated with Alcalase followed a slightly different order than for those treated with tryp- sin, as follows: FZ (43.98% ± 0.6 3% ) > HZ (36.93% ± 1.2%) > HMJ (33.86% ± 0.73%) > QL (22.84% ± 0.92%). These values were similar to those found in the literature; for instance, 20.8%-24.4% for Spirulina platensis hydrolyzed by Alcalase (Fan et al., 2018; WanG and zHanG, 2016). The peptide compositions of dark teas were analyzed by RP-HPLC. This study displays partial profiles during min 0-10, since no signal was obtained in the last 10 min (not included in Fig. 3). In min 0-10, obvious differences were seen between the complete protein extracts Fig. 1: Protein concentrations of four kinds of dark tea extracts and their hy- drolysates before and after purification treatments. Mean ± SD (n=3). Bars with different letters are significantly different at p < 0.05. Fig. 2: Degree of hydrolysis (DH, %) for four kinds of dark tea generated by trypsin and Alcalase and their complete protein extracts. Mean ± SD (n=4). Bars with different letters are significantly different at p < 0.05. 60 K. Su, X. Mao, L. Ai, X. Zhang Fig. 3: RP-HPLC absorbance profiles at 215 nm for complete proteins and hydrolysates of HMJ (a), FZ (b), HZ (c), and QL (d). and the trypsin- and Alcalase-generated hydrolysates. For example, the HMJ protein mainly had one wide peak at around 6 min, where- as after hydrolysis, there were two high peptide peaks. The trypsin hydrolysates displayed narrower and higher peaks than the Alcalase hydrolysates. All four kinds of dark tea hydrolysates generated by trypsin displayed similar, moderate peptide profiles in which the first peak was eluted out earlier, at around 5 min. The order of elution time was FZ < HMJ < HZ ≈ QL (Fig. 3), which was also consistent with the order of their DH values. This suggests that trypsin released peptides from the various dark teas at different rates. In addition, for both complete proteins and hydrolysates, a narrow high peak appeared at a retention time of 12.5 min, with a nearly undetectable signal at 280 nm (not included in Fig. 3), the absorbance of peptide bound. The strongest signal was at 215 nm, suggesting the presence of a flavanoid. Although for an individual protease, higher DH might cause more bioactive peptides to be released from proteins, the ultimate and maximal inhibition activity of the hydrolysates were determined by the enzyme’s structure, especially the nature of the peptides released in different hydrolysates. This explains the lack of correlation between the DH and the inhibitory activity of the samples. In vitro assessment of biological activity α-Amylase inhibition activity Several plant proteins and peptides have been found to inhibit α- amylase activity in vitro (SioW et al., 2017; SintSova et al., 2018; nataSHYa et al., 2018). However, to date, no reports are available on the α-amylase inhibitory activity of dark tea protein hydrolysates. The IC50 values of the four kinds of dark tea hydrolysates generated by trypsin and Alcalase were as follows: 2.78 ± 0.04 mg/mL for HMJ, 1.38 ± 0.03 mg/mL for FZ, 1.95 ± 0.06 mg/mL for HZ, and 1.27 ± 0.03 mg/mL for QL. The IC50 value of acarbose was 0.56 ± 0.06 mg/mL. It is possible that compounds with α-amylase inhibi- tory activity in the complete protein may be degraded during hydro- lysis by trypsin and Alcalase. A similar phenomenon was observed by HarnedY and FitzGerald (2013), in which the Palmaria Palmata protein exhibited higher renin inhibitory activity than its hydrolysate generated by Alcalase. Fig. 4 indicates the dose-response curves of four kinds of complete proteins. Inhibitory activity reached nearly its highest stage at a sam- ple concentration of 4 mg/mL, in the following order: QL (64.94% ± 1.25%) > FZ (61.08% ± 0.76%) > HZ (59.09% ± 1.10%) > HMJ (56.24% ± 0.90%). Interestingly, the α-amylase inhibitory activity of all four kinds of dark tea was largely diminished after hydrolysis, and none of the hydrolysate fractions demonstrated significant inhibitory activity at a concentration range of 1-4 mg/mL (indicated in Tab. 1). Most of the protein hydrolysates of HMJ, HZ, and QL negatively inhibited α-amylase. Low inhibitory activity was found in the hydrolysates of FZ; the highest value occurred at a concentration of 1 mg/mL, (21.91% ± 0.02% for the trypsin-generated hydrolysate and 29.48% ± 0.04% for the Alcalase-generated hydrolysate). No regular dose- response relationship was found (see Tab. 1). The reason for this is unknown; perhaps α-amylase’s acting sites are related to the cleav- age sites of trypsin and Alcalase, whereas FZ has a different protein composition than HMJ, HZ, and QL. Anti-diabetic potential of dark tea 61 α-Glucosidase inhibition activity As an important carbohydrate-hydrolyzing enzyme, α-glucosidase plays a key role in carbohydrate digestion. The inhibition of α- glucosidase helps to delay carbohydrate digestion and prolongs over- all carbohydrate digestion time, reducing the glucose absorption rate and consequently blunting the postprandial plasma glucose rise (bHandari et al., 2008; KiM et al., 2011). Several previous studies have reported the inhibitory activities of plants. For example, zHou et al. (2017) reported that flavanone compounds in dark tea had an IC50 value of 10.2 μM. liu et al. (2013) reported that aqueous ex- tracts prepared from Nelumbo nucifera leaves achieved an IC50 value of 1.86 ± 0.018 mg/mL. As indicated in Tab. 2, nearly all the proteins and their hydrolysates exhibited higher α-glucosidase inhibitory ac- tivity than acarbose (IC50 = 0.7265 ± 0.058 mg/mL). QL exhibited high activity, with an IC50 value of 0.0103 ± 0.025 mg/mL before hydrolysis, and 0.0349 ± 0.0025 mg/mL and 0.0233 ± 0.0024 mg/mL after treatment with trypsin and Alcalase, respectively. HMJ showed lower inhibitory capacity after being hydrolyzed, suggesting that some proteins with α-glucosidase inhibition activity may have been cleaved. Moreover, no significant differences were detected among the complete HZ proteins and the two enzymatic hydrolysates. The lowest inhibitory activity was observed in the FZ hydrolysate diges- ted by trypsin, with an IC50 value of 1.3114 ± 0.0174 mg/mL. DPP-IV inhibition activity The DPP-IV inhibitory activities of HMJ, FZ, HZ, and QL protein hydrolysates obtained by trypsin and Alcalase, respectively, were in- vestigated. In general, fairly high DPP-IV relative inhibitory activity was observed in the protein fractions, with HMJ having the largest DPP-IV relative inhibitory ability, followed by FZ and HZ; QL had the lowest DPP-IV relative inhibitory activity. In a study by HarnedY and FitzGerald (2013), an IC50 value of 2.52 ± 0.05 mg/mL was ob- tained for Palmaria palmata aqueous extracts generated by Alcalase, which is much higher than the value in the present study. The most effective protein fraction was HMJ hydrolyzed with Alcalase, which recorded an IC50 value of 0.1000 ± 0.0266 mg/mL. IC50 values of 0.1794 ± 0.0204 mg/mL and 0.1315 ± 0.0017 mg/mL were found in the complete HMJ protein and the trypsin-treated HMJ fraction. The relative inhibitory ability of FZ and HZ increased dramatically after hydrolysis (Tab. 2), suggesting that some effective bioactive peptides were obtained. QL appeared to have the least effective relative inhibi- tory activity but still had IC50 values of 1.0900 ± 0.027 mg/mL (con- trol), 1.3364 ± 0.0056 mg/mL (trypsin-generated hydrolysate), and 1.0281 ± 0.016 mg/mL (Alcalase-generated hydrolysate). The results of this in vitro study clearly suggest that dark tea proteins and their hydrolysates have the potential to control hyperglycemia. The α-amylase, α-glucosidase, and DPP-IV inhibitory activities ob- Fig. 4: Inhibitory activity against α-amylase of different concentrations of four kinds of complete black tea proteins. Mean ± SD (n=4). Tab. 1: Inhibitory activity (%) of four kinds of dark tea hydrolysates generated by trypsin(a) and Alcalase(b) against α-amylase at concentrations of 1 mg/mL, 2 mg/mL, and 4 mg/mL. Concentrations of 4–8 mg/mL were also investigated in the study but not included in this paper. Mean ± SD (n=3). n.a. = no inhibition detected. Values with different letters are significantly different at p < 0.05. Enzyme Sample Inhibitory activity (%) 4 mg/mL 2 mg/mL 1 mg/mL Trypsin HMJ -30.97 ± 0.06 n.a. -29.20 ± 0.04 n.a. -34.16 ± 0.06 n.a. FZ 7.88 ± 0.01 c 18.81 ± 0.02 b 21.91 ± 0.02 a HZ -26.44 ± 0.11 n.a. -4.66 ± 0.10 n.a. 2.36 ± 0.05 d QL -5.91 ± 0.02 n.a. -10.45 ± 0.03 n.a. -2.65 ± 0.05 n.a. Alcalase HMJ -30.51 ± 0.04 n.a. -28.81 ± 0.03 n.a. -20.49 ± 0.08 n.a. FZ 1.68 ± 0.03 c 14.84 ± 0.13 b 29.48 ± 0.04 a HZ -54.44 ± 0.08 n.a. -51.27 ± 0.07 n.a. -24.21 ± 0.02 n.a. QL -7.96 ± 0.05 n.a. -13.30 ± 0.01 n.a. -3.10 ± 0.01 n.a. Tab. 2: IC50 (concentration that inhibits enzyme activity by 50%) of four kinds of dark tea and their hydrolysates generated by trypsin and Alcalase against α-glucosidase and DPP-Ⅳ. Mean ± SD (n=3). IC50 with different letters for each of the activities are significantly dif- ferent at p < 0.05. Sample Hydrolysate IC50 (mg/mL) for α-glucosidase Complete Trypsin Alcalase HMJ 0.0942 ± 0.0023 g 0.2161 ± 0.0036 f 0.2036 ± 0.0022 f FZ 0.5401 ± 0.0042 d 1.3114 ± 0.0174 a 0.6588 ± 0.0045 b HZ 0.5478 ± 0.0128 d 0.6231 ± 0.0355 c 0.5069 ± 0.0011 e QL 0.0103 ± 0.0025 h 0.0349 ± 0.0026 h 0.0233 ± 0.0024 h Sample Hydrolysate IC50 (mg/mL) for DPP-Ⅳ Complete Trypsin Alcalase HMJ 0.1794 ± 0.0204 ghi 0.1315 ± 0.0017 hi 0.1000 ± 0.0266 i FZ 0.9124 ± 0.0216 d 0.1011 ± 0.0190 i 0.1998 ± 0.0176 gh HZ 0.8467 ± 0.0035 e 0.3993 ± 0.0178 f 0.2477 ± 0.0278 g QL 1.0900 ± 0.0270 b 1.3364 ± 0.0056 a 1.0281 ± 0.0160 c 62 K. Su, X. Mao, L. Ai, X. Zhang DOI: 10.1016/j.foodchem.2012.08.038 KiM, J.S., HYun, t.K., KiM, M.J., 2011: The inhibitory effects of ethanol ex- tracts from sorghum, foxtail millet and proso millet on α-glucosidase and α-amylase activities. Food Chem. 124 (4), 1647-1651. DOI: 10.1016/j.foodchem.2010.08.020 KoH, l.W., WonG, l.l., loo, Y.Y., KaSaPiS, S., HuanG, d., 2010: Evaluation of different teas against starch digestibility by mammalian glycosidases, J. Agric. Food Chem. 58(1), 148-154. DOI: 10.1021/jf903011g lebovitz, H.e., 1997: Alpha-glucosidase inhibitors. Endocrinol. Metab. Clin. North Am. 26, 539-551. liu, S., li, d., HuanG, b., CHen, Y., lu, X., 2013: Inhibition of pancreatic lipase, α-glucosidase, α-amylase, and hypolipidemic effects of the total flavonoids from Nelumbo nucifera leaves. J. Ethnopharmacol. 149, 263- 269. lin, Y.S., CHen, C.r., Wu, W.H., Wen, C.l., CHanG, C.i., Hou, W.C., 2015: Anti-α-glucosidase and Anti-dipeptidyl Peptidase-IV Activities of Extracts and Purified Compounds from Vitis thunbergii var. taiwaniana. J. Agric. Food Chem. 63(28), 6393-6401. DOI: 10.1021/acs.jafc.5b02069 Miao, M., JianG, b., JianG, H., zHanG, t., li, X.. 2015: Interaction mecha- nism between green tea extract and human alpha-amylase for reducing starch digestion, Food Chem. 186, 20-25. DOI: 10.1016/j.foodchem.2015.02.049 PenG, S., Xue, l., lenG, X., YanG, r., zHanG, G., HaMaKer, b.r., 2015: Slow digestion property of octenyl succinic anhydride modified waxy maize starch in the presence of tea polyphenols. J. Agric. Food Chem. 63 (10), 2820-2829. li, S., lo, C.Y., Pan, M.H., lai, C.S., Ho, C.t., 2012: Black tea: chemical analysis and stability. Food Funct. 4(1), 10-18. DOI: 10.1039/C2FO30093A SintSova, o., GladKiKH, i., CHauSova, v., MonaStYrnaYa, M., anaStYuK, S., CHerniKov, o., YurCHenKo, e., aMinin, d., iSaeva, M., leYCHenKo, e., KozlovSKaYa, e., 2018: Peptide fingerprinting of the sea anemone Heteractis magnifica mucus revealed neurotoxins, Kunitz-type proteinase inhibitors and a new β-defensin α-amylase inhibitor. J. Proteomics. 173, 12-21. DOI: 10.1016/j.jprot.2017.11.019 SioW, H.l., Gan, C.Y., 2016: Extraction, identification, and structure-activity relationships of antioxidative and α-amylase inhibitory peptides from cumin seeds (Cuminum cyminum). J. Funct. Foods 22, 1-12. DOI: 10.1016/j.jff.2016.01.011 SioW, H., liM, t.S., Gan, C., 2017: Development of a workflow for screen- ing and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study − Cumin seed. Food Chem. 214, 67-76. DOI: 10.1016/j.foodchem.2016.07.069 SPellMan, d., MCevoY, e., FitzGerald, r.J., 2003: Proteinase and exo- peptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. Int. Dairy J. 13(6), 447-453. DOI: 10.1016/S0958-6946(03)00053-0 Stote, K.S., baer, d.J., 2008: Tea consumption may improve biomarkers of insulin sensitivity and risk factors for diabetes. J. Nutr. 138(8), 1584- 1588. WanG, z.J., zHanG, X.W., 2016: Inhibitory effects of small molecular pep- tides from Spirulina (Arthrospira) platensis on cancer cell growth. Food Funct. 7, 781-788. WanG, z.J., zHanG, X.W., 2017: Isolation and identification of anti-proli- ferative peptides from Spirulina platensis using three-step hydrolysis. J. Sci. Food Agric. 97, 918-922. Xiao, J., Huo, J., JianG, H., YanG, F., 2011: Chemical compositions and bio- activities of crude polysaccharides from tea leaves beyond their useful date. Int. J. Biol. Macromol. 49(5), 1143-1151. Yu, z., Yin, Y., zHao, W., WanG, F., Yu, Y., liu, b., liu, J., CHen, F., 2011a: Characterization of ACE-inhibitory peptide associated with antioxidant and anticoagulation properties. J. Food Sci. 76(8), 1149-1155. DOI: 10.1111/j.1750-3841.2011.02367.x Yu, z., Yin, Y., zHao, W., Yu, Y., liu, b., liu, J., CHen, F., 2011b: Novel served were different for HMJ, FZ, HZ, and QL proteins and their hydrolysates generated by trypsin and Alcalase. Generally, all four kinds of dark tea protein extracts demonstrated moderate inhibitory activity on α-amylase (IC50 values ranged from 1.27 to 2.78 mg/mL), but inhibitory activity was nearly undetectable after hydrolysis. All of the proteins and hydrolysates displayed good inhibitory activity on α-glucosidase (IC50 values ranged from 0.0103 to 1.3114 mg/mL) and DPP-IV (IC50 values ranged from 0.1000 to 1.3364 mg/mL). The HMJ and QL extracts were better α-glucosidase inhibitors, while the HMJ extracts, FZ hydrolysates generated with trypsin and Alcalase, and HZ hydrolysates generated with trypsin and Alcalase recorded better inhibitory activity on DPP-IV than other fractions. It is noted that the protein concentrations in dark tea under typical drinking conditions (steeped at 85 ℃ for 10 min) detected by Brad- ford assay were as follows: 0.03595 mg/mL (HMJ), 0.04758 mg/mL (FZ), 0.01493 mg/mL (HZ), and 0.08977 mg/mL (QL). Thus, theo- retically, the protein concentrations in tea as it is typically drunk is close to the IC50 value for α-glucosidase, but 10- and 100-fold con- centrations of the tea, respectively, would be required to achieve the IC50 values for DPP-IV and α-amylase. In addition, due to the degra- dation of protein or peptides in the gastrointestinal tract, doses higher than the theoretical values are needed; hence, more effective protein- extraction techniques deserve further study in the future. References bHandari, M.r., nilubon, J.a., HonG, G., KaWabata, J., 2008: α- Glucosidase and α-amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliata, Haw.). Food Chem. 106, 247-252. DOI: 10.1016/j.foodchem.2007.05.077 CHen, G., Guo, M., 2017: Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration–HPLC-MS. Front. Pharma- col. 8, 1-8. DOI: 10.3389/fphar.2017.00228 CHen, d., Sun, J., donG, W., SHen, Y., Xu, z., 2018: Effects of polysac- charides and polyphenolics fractions of Zijuan tea (Camellia sinensis var. kitamura) on α-glucosidase activity and blood glucose level and glucose tolerance of hyperglycaemic mice. Int. J. Food Sci. Technol. 53, 2335- 2341. Fan, X.d., Cui, Y.J., zHanG, r.l., zHanG, X.W., 2018: Purification and identification of anti-obesity peptides derived from Spirulina platensis. J. Funct. Foods 47, 350-360. DOI: 10.1016/j.jff.2018.05.066 Gao, r., WanG, Y. , Wu, z., MinG, J., zHao, G., 2012: Interaction of Barley β-Glucan and Tea Polyphenols on Glucose Metabolism in Streptozotocin- Induced Diabetic Rats. J. Food Sci. 77, H128-H134. DOI: 10.1111/j.1750-3841.2012.02688.x Green, b.d., Gault, v.a., o’Harte, F.P., Flatt, P.F., 2004: Structurally modified analogues of glucagon-like peptide-1 (GLP-1) and glucose- dependent insulinotropic polypeptide (GIP) as future antidiabetic agents. Curr Pharm Des. 10, 3651-3662. Gron, H., Meldal, M., breddaM, K., 1992: Extensive comparison of the substrate preferences of two subtilisins as determined with peptide sub- strates which are based on the principal of intramolecular quenching. Bio- chem. 31, 6011-6018. HarnedY, P.a., FitzGerald, r.J., 2013: In vitro assessment of the cardio- protective, anti-diabetic and antioxidant potential of Palmaria palmata protein hydrolysates. J. Appl. Phycol. 25(6), 1793-1803. DOI: 10.1007/s10811-013-0017-4 HuanG, Q., CHen, S., CHen, H., WanG, Y., WanG, Y., HoCHStetter, d., Xu, P., 2013: Studies on the bioactivity of aqueous extract of pu-erh tea and its fractions: in vitro antioxidant activity and α-glycosidase inhibitory property, and their effect on postprandial hyperglycemia in diabetic mice. Food Chem. Toxicol. 53, 75-83. DOI: 10.1016/j.fct.2012.11.039 KalYanKar, P., zHu, Y., o’KeeFFe, M., o’Cuinn, G., FitzGerald, r.J., 2013: Substrate specificity of glutamyl endopeptidase (GE): hydrolysis studies with a bovine α-casein preparation. Food Chem. 136, 501-512. Anti-diabetic potential of dark tea 63 peptides derived from egg white protein inhibiting alpha-glucosidase. Food Chem. 129(4), 1376-1382. DOI: 10.1016/j.foodchem.2011.05.067 Yu, z., Yin, Y., zHao, W., 2012: Anti-diabetic activity peptides from albumin against α-glucosidase and α-amylase. Food Chem. 135(3), 2078-2085. DOI: 10.1016/j.foodchem.2012.06.088 zHou, H., lia, H., YanG, M., 2017: C-geranylated flavanones from YingDe dark tea and their antioxidant and α-glucosidase inhibition activities. Food Chem. 235, 227-233. Address of the corresponding author: Prof. X.W. Zhang, College of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640 E-mail: snow_dance@sina.com © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en).