Alpha-glucosidase inhibitory effects of propolis extracts in
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Alpha-glucosidase inhibitory effects of propolis extracts in
1 Inhibitory properties of aqueous ethanol extracts of 2 propolis on alpha-glucosidase 3 Hongcheng Zhang , Guangxin Wang , Trust Beta , Jie Dong1,3* 4 (1 Bee Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100093, 5 China; 2 Department of Food Science, University of Manitoba, Winnipeg, R3T 2N2, 6 Canada; 3 National Research Center of Bee Product Processing, Ministry of Agriculture, 7 Beijing, 100093, China) 1,3 1 2 8 9 10 *Corresponding author: Tel.: +86 10 62594264; Fax: +86 10 62594264 11 Email: jiedon@126.com (J. Dong) 12 Address: Bee Research Institute, 13 Chinese Academy of Agricultural Sciences, 14 Xiangshan, Beijing, 100093, China 15 16 17 1 18 Abstract 19 The objective of the present study was to evaluate the inhibitory properties of various 20 extracts of propolis on alpha-glucosidase from baker’s yeast and mammalian intestine. 21 Inhibitory activities of aqueous ethanol extracts of propolis were determined by using 22 4-nitrophenyl-D-glucopyranoside, sucrose and maltose as substrates, and acarbose as 23 a positive reference. All extracts were significantly effective in inhibiting 24 α-glucosidase from baker’s yeast and rat intestinal sucrase in comparison with 25 acarbose (p<0.05). The 75% ethanol extracts of propolis (75% EEP) showed the 26 highest inhibitory effect on α-glucosidase and sucrase and was a noncompetitive 27 inhibition mode. 50% EEP, 95% EEP and 100% EEP exhibited a mixed inhibition 28 mode; while water extracts of propolis (WEP) and 25% EEP demonstrated a 29 competitive inhibition mode. Furthermore, WEP presented the highest inhibitory 30 activity against maltase. These results suggest that aqueous ethanol extracts of 31 propolis may be used as nutracueticals for the regulation of postprandial 32 hyperglycemia. 33 34 Key Word: Propolis; Alpha-glucosidase; Inhibitory properties; Postprandial 35 hyperglycemia 36 37 38 39 2 40 1 Introduction 41 Diabetes mellitus is the most common endocrine disorder disease caused by 42 inherited or acquired deficiency in insulin excretion and by decreased responsiveness 43 of the organs to secreted insulin. According to the recent reports, the number of 44 diabetic patients in the world would amount to 366 million in 2030 and death related 45 to diabetes mellitus account for about 9% global mortality, particularly type 2 diabetes 46 mellitus (Adolfo, Jaime, & René, 2008). In China, more than 92 million adults have 47 diabetes, and 95 percent of them are type 2 diabetes mellitus, according to the survey 48 of current epidemiology (Dai & Wang, 2011). Because of more-affluent lifestyles, the 49 number of people with diabetes is predicted to increase in the future. Patients with 50 type 2 diabetes mellitus usually tend to have long-term complications, such as 51 retinopathy, cataract, atherosclerosis, neuropathy, nephropathy and impaired wound 52 healing (Ahmed, 2005). Diabetes mellitus is characterized by abnormally high plasma 53 glucose. Hyperglycemia has played a central role in pathogenesis of complications 54 related to diabetes mellitus. In treatment of type 2 diabetes, suppression of 55 postprandial hyperglycemia can decrease the risk of those complications. Hence, 56 control of postprandial hyperglycemia should be a primary goal in the prevention and 57 management of type 2 diabetes. 58 Alpha-glucosidases are a series of enzymes, including sucrase and maltase, 59 located in the brush-border surface of intestinal cell, which catalyze the final step in 60 the digestive process of carbohydrates to release absorbable monosaccharides 61 resulting in increased blood glucose levels (Shai et al., 2010). If α-glucosidases are 3 62 inhibited, the liberation of D-glucose from dietary complex carbohydrates can be 63 retarded. Thus, α-glucosidase inhibitors have become candidates of hot pursuits to 64 delay the digestion and absorption of carbohydrates and restrain postprandial 65 hyperglycemic excursions. Alpha-glucosidase inhibitors as one of therapeutic 66 approaches for diabetes mellitus have been known since the early 1990s (Ye, Shen, & 67 Xie, 2002). At present, some α-glucosidase inhibitors, such as acarbose, miglitol and 68 voglibose, have been approved for clinical use in the management of type 2 diabetes, 69 as well as the treatment of obesity. However, some synthetic α-glucosidase inhibitors 70 have side-effects, such as flatulence, diarrhea and abdominal cramping, all of which 71 are associated with incomplete carbohydrate absorption (Ghadyale, Takalikar, 72 Haldavnekar, & Arvindekar, 2012). As a result, many researchers have focused on 73 novel α-glucosidase inhibitors from natural materials, which are used to develop 74 functional foods or lead compounds for antidiabetic treatment including Syagrus 75 romanzoffiana, Adhatoda vasica Nees, Syzygium cumini (Linn.) seed kernel (Matsui, 76 et al, 2001; Murai, et al, 2002; Shinde, et al, 2008). Some medicinal plant species 77 have more potent α-glucosidase inhibitory activities than powerful synthetic 78 α-glucosidase inhibitors such as acarbose (Andrade, Becerra, & Cárdenas, 2008). 79 Propolis is a colored and aromatic colloidal substance collected by honeybees 80 through adding their saliva secreted to the resinous plant exudate, which is used to 81 build honeycomb and to fight against the invasion of pathogenic microorganism. The 82 chemical composition of propolis varies and depends mainly upon the local flora in 83 the region of collection. At present, more than 300 components have been identified 4 84 including flavonoids, phenolic acids, alcohols and their esters, ketones, and inorganic 85 compounds (Banskota, Tezuka, & Kadota, 2001). Propolis has been applied in popular 86 folk medicine since 3000 BC due to possessing a broad spectrum of biological 87 activities such as antioxidant, antimicrobial, anti-inflammatory, antiviral, anticancer 88 and antihepatotoxic properties (Bankova, 2005). Recent studies show that ethanol and 89 water extracts of propolis can control the glycemia and modulate glucose and lipid 90 metabolism in STZ-induced diabetic rats (Hu, et al.,2005; Yan, et al. 2008). Ethanolic 91 extracts of Brazilian green propolis were also shown to possess therapeutic potential 92 in STZ-induced diabetic rats (Búfalo et al., 2009). In China, propolis has been 93 approved for use in functional foods carrying a health claim of controlling glycemia in 94 1999 by the Ministry of Health (State Administration of Traditional Chinese Medicine, 95 1999). Moreover, propolis has been accepted as adjuvant therapy drugs for diabetes 96 mellitus in Chinese Pharmacopoeia in 2005 (State Pharmacopoeia Commission, 2005). 97 However, there are few reports whether controlling glycemia of propolis is related to 98 its inhibitory activities against α-glucosidase. 99 In the present study, we assessed inhibitory effects of aqueous ethanol extracts of 100 propolis on alpha-glucosidase and the kinetics of enzyme inhibition by using the 101 Michaelis-Menten model. 102 2 Material and Methods 103 2.1 Chemicals and reagents 104 Alpha-glucosidases (EC 3.2.1.20) from baker’s yeast, rat intestinal acetone powder, 105 p-Nitrophenyl-α-D-glucopyranoside (PNP-glucoside), Sucrose, Maltose, 106 Piperazine-1,4-bisethanesulfonic acid (PIPES), Folin-Ciocalteu, and Glucose-Trinder 5 107 100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl 108 sulphoxide (DMSO, cell culture grade) was purchased from Applichem Co. 109 (Darmstadt, Germany). The crude propolis was procured from Bee Research Institute, 110 Chinese Academy of Agricultural Science (Beijing, China). All reagents were of 111 analytical grade. 112 2.2 Preparation of aqueous ethanol extracts of propolis 113 Proplis extracts were prepared as described by Huang et al. (2009) with slight 114 modification. Crude propolis (10 gram) was respectively extracted with 200ml 115 aqueous ethanol solvent at concentrations ranging from 0%, 25%, 50%, 75%, 95% to 116 100% (in water, v/v) by using ultrasonic extract for 4 hours (ultrasonic extractor, 117 DCTZ-1000, Beijing Hongxianglong Biotechnol Co. Ltd., Beijing, China). The 118 suspensions were centrifuged at 12,000×g for 30 min to obtain supernatants which 119 were concentrated at 45℃ in a rotary evaporator (Rotavapor, R-215, Buchi Co., Ltd, 120 Switzerland) and then freeze-dried. Extracts were stored in ziplock bag at 4℃ in 121 darkness. The extracts were dissolved by DMSO until application. 122 2.3 Determination of total phenolic contents 123 Total polyphenol contents in propolis extracts were determined according to the 124 Folin-Ciocalteu colorimetric method with slight modifications (Kumazawa, et al., 125 2002). A standard curve was built with gallic acid solution. Aliquots ranging from 0 to 126 1.2 ml of standard solution (100 µg/ml) were pipetted into 10 ml volumetric flasks 127 containing 6 ml distilled water. 0.5 ml of 2 mol/L Folin-Ciocalteu solution and 1.5 ml 128 of 10% Na2CO3 (w/v) were added and the volume made up to 10 ml with distilled 6 129 water. Following mixing, the solution was measured at 765nm after 10min by using 130 UV-2500/Ultraviolet visible spectrophotometer with UVProbe software (Shimadzu 131 Co., Ltd., Tokyo, Japan). The blank was also prepared without addition of the 132 standard aliquots. For determination of the total phenolic contents in propolis extracts, 133 20μl of aqueous solution at a concentration of 1 mg/ml was used. Total phenolic 134 contents were expressed as milligrams gallic acid equivalents per gram extract (mg 135 GAE / g). 136 2.4 Determination of total flavonoid contents 137 Contents of flavonoid in propolis extracts were determined according to the 138 method of Kaijv, Sheng, and Chao (2006), with minor modifications. A standard curve 139 was built with chrysin solution. Aliquots ranging from 0 to 12 ml of standard solution 140 (100 µg/ml) were pipetted into 25 ml volumetric flasks containing 1.0 ml aliquots of 141 5% NaNO2 (w/v) solution. After 6 min, 1.0 ml 10% AL(NO3)3 (w/v) solution was 142 added and thoroughly mixed. 10ml 4.3% NaOH (w/v) solution were added after 6min 143 and the volume was made up with ethanol. The blank was prepared by using ethanol 144 instead of standard solution or sample solutions. After 15 min, the absorbance was 145 measured at 510 nm by using UV-2500/Ultraviolet visible spectrophotometer with 146 UVProbe software (Shimadzu Co., Ltd., Tokyo, Japan). For determination of the total 147 flavonoid contents in propolis extracts, 0.5 ml of aqueous solution at a concentration 148 of 1mg/ml were used. Total flavonoid contents were expressed as milligramms 149 chrysin equivalents of per gram extract (CE). 150 2.5 Inhibitory activity assay for baker’s yeast alpha-glucosidase 7 151 Alpha-glucosidase inhibitory effect of propolis extracts was assayed according to 152 the procedure described previously by Kim et al. (2005) with slight modifications. 153 The enzyme reaction was performed using PNP-glycoside as a substrate in 0.1M 154 PIPES buffer (pH 6.8). 1ml PNP-glycoside (1.0 mM) was premixed with 0.1 ml 155 propolis extracts at various concentrations (2μg/ml-200μg/ml) in 2.05ml PIPES buffer 156 and the reaction system was preheated at 37oC for 30 min. Then, 0.5 ml enzyme 157 solution (0.11 U/ml) was added into mixture and absorbances were immediately 158 measured at 30 second intervals for 15 min at 415nm using UV-2500/Ultraviolet 159 visible spectrophotometer with UVProbe software (Shimadzu Co., Ltd., Tokyo, Japan). 160 The inhibition activity was calculated by the equation: Inhibition (%) = [1-(A 161 sample/A control)] ×100% (Kim et al. 2005). Acarbose was also assayed as a positive 162 reference. Concentrations of extracts resulting in 50% inhibition of enzyme activity 163 (IC50 values) were determined graphically. Different propolis extracts were compared 164 on the basis of their IC50 values estimated from the dose response curves. 165 2.6 Inhibitory activity assay for rat intestinal sucrase 166 Rat intestinal sucrase inhibitory effects of propolis extracts was assayed 167 according to a procedure described previously (Nishioka et al., 1998) with slight 168 modifications. Briefly, 100 mg rat intestinal acetone powder was homogenized in 15 169 ml 0.9 % NaCl solution (w/v). After centrifugation at 12,000g for 40 min, the 170 supernatant was stored at 4oC as enzyme solution. 0.8 ml 56mmol/L Sucrose and 0.75 171 ml 0.1 M PIPES buffer (pH 7.0) were premixed with 0.1ml propolis extracts of 172 various concentrations (2μg/ml-200μg/ml). After pre-incubating for 5 min at 37 oC, 8 173 0.7 ml of the enzyme solution was added and the reaction was carried out at 37oC for 174 30 min. The reaction was terminated by adding 0.75 ml of 2 mol/L Tris-HCl buffer 175 (pH 6.9). For the blank, 0.1 ml DMSO was used in place of propolis extract. The 176 concentration of glucose released from the reaction mixtures was determined 177 colorimetrically using Glucose-Trinder 100 (Hwang et al., 2000) and the absorbance 178 measured at 505 nm after incubation at 37oC for 20 min. The inhibition activity was 179 calculated by the equation: Inhibition (%) = [1-(A sample/A control)] ×100%. 180 Acarbose was also assayed as a positive reference. 181 2.7 Inhibitory activity assay for rat intestinal maltase 182 Rat intestinal maltase inhibitory effect of propolis extracts was assayed 183 according to a procedure described previously (Toda, Kawabata and Kasai, 2000). The 184 assay for rat intestinal maltase inhibitory activity was carried out in a similar manner 185 as the rat intestinal sucrase inhibitory activity, except that 10mM maltose in 0.1M 186 PIPES buffer (pH 7.0) was used as a substrate. Acarbose was also assayed as a 187 positive reference. 188 2.8 Inhibition kinetics on alpha-glucosidase activity 189 Inhibition model of different extracts of propolis on baker yeast’s α-glucosidase 190 was tested according to procedure described previously (Shai et al., 2010) with minor 191 modifications. Alpha-glucosidase activity was measured with increasing 192 concentrations of PNP-glucoside (0.4-6.0 mM) in the absence or presence of propolis 193 extracts at various concentrations (0.1-2.0 mg/ml). The reaction was initiated by 194 addition of enzyme and the reaction measured at 415 nm at 30 seconds interval for 15 9 195 min. The enzyme assay data containing various concentration of PNP-glucoside and 196 propolis extracts were used to construct the Lineweaver-Burk plots to determinate 197 inhibition model, Vmax and Km values. 198 2.9 Inhibition constant analysis The inhibition constant of propolis on alpha-glucosidase was calculated 199 200 by Ki [I ] α 1 , where [I] represents the concentration of inhibitor, and α is confirmed v V max/α [ S ] Km [ S ] . Vmax and Km values were obtained from the above assay, and [S] 201 by 202 represents the concentration of substrate. 203 2.10 Statistical analysis 204 Experimental results were expressed as mean ± standard deviation (SD) of 205 triplicate measurements. The data were subjected to one way analysis of variance 206 (ANOVA). Significance differences at p<0.05 among treatment means were obtained 207 using Duncan’s multiple range test using SAS version 9.1 (SAS Institute Inc., Cary, 208 NC, USA). 209 3 Results and Discussion 210 3.1 Total phenolic and flavonoid contents of aqueous ethanol extracts of propolis 211 Phenolics are the predominant bioactive materials in propolis which have been 212 reported to have multiple biological effects, including antidiabetes. Therefore, 213 measurement of total phenolic contents (TPC) and total flavonoid contents (TFC) was 214 inevitable. Total phenolic and flavonoid contents in various aqueous ethanol extracts 215 of propolis are presented in Table 1. TPC ranged from 273.94 to 386.49 mgGAE/g 216 extracts increasing in the following order: 25% EEP > WEP > 50% EEP > 75% EEP > 10 217 95% EEP > 100% EEP. TPC was not significantly different among WEP, 25% EEP, 218 and 50% EEP. TFC ranged from 352.32 to 697.36 mg CE/g extracts increasing in the 219 following order: 75% EEP > 100% EEP > 95% EEP > 50% EEP > 25% EEP > WEP. 220 TFC was not significantly different among 100% EEP, 95% EEP, and 75% EEP. 221 The total phenolic and flavonoid contents of propolis extracts varied with 222 different concentrations of hydrous ethanol. A similar report shows that ethanol/water 223 concentrations correlates with the amount and composition of phenolic compounds 224 and flavonoids of propolis extracts (Cottica et al. 2011). Moreover, propolis from 225 various areas of China was found to contain a wide variety of bioactive compounds, 226 mainly phenolic acids and flavonoids (Zhou et al., 2008). In the current study, while 227 ethanol concentrations in hydrous ethanol were less than 50% as extraction solvent, 228 the TPC of these extracts were significantly higher than those containing higher 229 ethanol concentrations (p<0.05). These propolis extracts may mainly contain more 230 hydrophilic phenolic compounds, cinnamic acid derivatives (Nakajima et al, 2007). 231 On the other hand, when ethanol concentrations were higher than 50%, TFC of the 232 extracts were significantly higher compared to those with lower ethanol 233 concentrations (p<0.05). These propolis extracts mainly contain a significant increase 234 in the ratio of more hydrophobic flavonoid compounds, such as apigenin, kaempferol 235 and chrysin (Zhou et al., 2008). 236 3.2 Inhibition of aqueous ethanol extracts of propolis against alpha-glucosidase 237 The α-glucosidase inhibitory activity of various propolis extracts is shown in table 238 2. The propolis extracts had ever been found to be reversibly bound to α-glucosidase 11 239 in our previous report. The effectiveness of enzymatic inhibition of the various 240 extracts was determined by calculating IC50. The lower of the value showed the higher 241 of the enzymatic inhibition. IC50 values of propolis extracts ranged from 7.24 to 20.01 242 μg/ml against baker’s yeast α-glucosidase and from 32.34 to 53.12 μg/ml against rat 243 intestinal sucrase. These values were significantly (p<0.05) lower than 177.5 μg/ml 244 and 538.3μg/ml of acarbose, a positive reference, respectively. Thus, all propolis 245 extracts possessed much stronger inhibitory effects on α-glucosidase from baker’s 246 yeast and sucrase compared to acarbose. The 75% EEP showed the highest inhibitory 247 effect on α-glucosidase from baker’s yeast and sucrase, and IC50 values accounted for 248 only 4% and 6% of that of acarbose, respectively. For rat intestinal maltase, WEP had 249 the highest inhibitory activity among all extracts with IC50 value of 32.67 μg/ml that 250 was significantly higher than that of acarbose of 22.5μg/ml (p<0.05). All propolis 251 extracts showed weak inhibitory effects on maltase in comparison to acarbose. 252 Many plant extracts from food and Chinese traditional medicine have been 253 reported to have anti-diabetic activity (Ye, Shen, Xie 2002). These anti-diabetic 254 phytochemicals are probably comprising phenolic compounds, such as flavonoids and 255 phenolic acids (Tadera, Minami, Takamatsu, & Matsuoka, 2006). Propolis extracts 256 contain phenolic compounds which are classified into two major categories, phenolic 257 acids and flavonoids. As shown in table 1, TPC and TFC of various ethanol extracts of 258 propolis were different. Similarly, the inhibitory effects of various propolis extracts on 259 alpha-glucosidases were also different (Table 2). The 75% EEP possessed the highest 260 flavonoid contents and the strongest inhibitory effect on α-glucosidases from baker’s 12 261 yeast and rat intestinal sucrase among all extracts. Wang, et al. (2010) also reported 262 that some flavonoids have the higher inhibitory effect on rat sucrase than that of rat 263 maltase. Moreover, the study revealed WEP and 25% EEP had higher TPC and 264 stronger inhibitory effects on rat intestinal maltase among all extracts. Some phenolics 265 such as chebulanin, chebulagic acid and chebulinic acid were reported to have the 266 same potential against rat intestinal maltase (Kumar et al., 2011). Kamiyama (2010) 267 similarly found that some catechin derivatives had better inhibitory activity against rat 268 intestinal maltase than rat intestinal sucrase. Therefore, it seems to assume that 269 extracts with higher TFC could have better inhibition against rat intestinal sucrase; 270 similarly extracts with higher TPC could possess better inhibition against rat intestinal 271 maltase. 272 3.3 Inhibitory kinetics of aqueous ethanol extracts of propolis against baker’s 273 yeast alpha-glucosidase 274 Yeast α-glucosidase is readily available in a pure form and has been widely used 275 for anti-diabetic nutraceutical and medicinal investigations as a model for screening 276 potential inhibitors and studying inhibitory mechanism (Ye, Shen, & Xie, 2002). To 277 find the inhibition mechanism of aqueous ethanol extracts of propolis, inhibitory 278 kinetics against yeast α-glucosidase was analyzed by Lineweaver-Burk plots using 279 data derived from enzyme assay containing different concentrations of PNP-glucoside 280 in the absence or presence of different concentration of inhibitor. 281 As can be seen in Table 3, various aqueous ethanol extracts of propolis had 282 different inhibition modes. Double-reciprocal plots of enzyme kinetics demonstrated 13 283 that the inhibition of WEP and 25% EEP was competitive inhibition mode (Fig.1A 284 and B), and the Ki values were 10.43μg/ml and 9.85µg/ml (Table 3). Plots also 285 indicated that the type of 50% EEP, 95% EEP and 100% EEP was mixed inhibition 286 mode (Fig.1C, E and F), and the Ki values were 9.92 μg/ml, 9.65 μg/ml and 10.89 287 μg/ml, respectively (Table 3). Moreover, inhibitory mode of 75% EEP was 288 noncompetitive inhibition (Fig.1D), and the Ki value was 15.18 μg/ml, a value 289 significantly higher than those of other extracts (p<0.05). 290 Phenolic compounds are able to inhibit the activities of carbohydrate-hydrolysing 291 enzymes due to their ability to bind with proteins (Shobana, Sreerama, & Malleshi, 292 2009). As can be seen, different aqueous ethanol extracts of propolis were revealed to 293 have different inhibition modes against α-glucosidase. Probably that is because 294 phenolic compounds in different EEP have different bound modes with α-glucosidase. 295 The inhibition of WEP and 25% EEP was a competitive mode characterized by the 296 fact that the substrate and inhibitor compete for the same binding site in the enzyme, 297 the so-called active site or substrate-binding site. It indicated that phenolic compounds 298 in WEP and 25% EEP can bind to the active site of α-glucosidase. The inhibition of 299 75%EEP was a noncompetitive mode in that inhibitor is not binding to the active site 300 on α-glucosidase. Inhibition mode was a mixed inhibition for 50%EEP, 95%EEP and 301 100%EEP. For the noncompetitive and mixed inhibition mode, the binding of 302 inhibitor can influence the binding of substrate by changing the conformation of the 303 enzyme. Different inhibition modes shown by various EEP were likely due to the 304 different bioactive compounds in the extracts. 75% EEP was found to contain the 14 305 highest TFC among EEP and exhibit non-competitive inhibition. TFC of 95%EEP and 306 100% EEP was lower but not significantly different than 75% EEP and showed mixed 307 inhibition. A similar trend was observed that many flavonoids exhibited mixed or 308 noncompetitive type of inhibition (Tadera, Minami, Takamatsu, & Matsuoka, 2006; 309 Xu, 2010). WEP and 25%EEP contained the higher TPC and exhibited a competitive 310 inhibition mode. The ethanol extract of G. montanum rich in phenolic composition 311 also showed competitive inhibition against yeast α-glucosidase (Ramkumar, 2010). It 312 seems to assume that inhibition of aqueous ethanol extracts of propolis with the higher 313 TPC is likely a competitive mode while those with higher TFC tend to have a 314 noncompetitive or mixed mode. 315 All aqueous ethanol extracts of propolis showed stronger inhibition against the 316 yeast α-glucosidase compared to acarbose, especially for those extracts using the 317 higher concentration of ethanol as the solvent. It is probably that the ability to bind to 318 wide regions of enzyme other than the active site enables these propolis extracts as 319 noncompetitive or mixed inhibitors a broader specificity of inhibition, compared with 320 acarbose, a competitive inhibitor. Another advantage of aqueous ethanol extracts of 321 propolis over acarbose is that these propolis extracts, noncompetitive or mixed 322 inhibitors, may not be affected by the higher concentration of the substrate in contrast 323 to acarbose which is a competitive inhibitor. It is reported that, with higher 324 carbohydrate food intake, higher concentrations of acarbose as a competitive inhibitor 325 would be needed to show the same effect. For mixed inhibition, the inhibitor would be 326 still effective at lower concentrations (Ghadyale et al., 2012). 15 327 328 4. Conclusion In the present study, all aqueous ethanol extracts of propolis show stronger 329 inhibitory effects on yeast α-glucosidase and rat intestinal sucrose than that of 330 acarbose as a positive reference. 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Geographical 437 traceability of propolis by high-performance liquid-chromatography fingerprints. 438 Food Chemistry, 108, 749-759. 439 20 440 Table 1 Total phenolic and flavonoid contents of various ethanol extracts of propolis Aqueous ethanol extracts of propolis (EEP) Water extracts of propolis (WEP) 25% ethanol extracts of propolis (25% EEP) 50% ethanol extracts of propolis (50% EEP) 75% ethanol extracts of propolis (75% EEP) 95% ethanol extracts of propolis (95% EEP) 100% ethanol extracts of propolis (100% EEP) Total phenolic contents (mg Total flavonoid contents (mg GAE/g extracts) CE/g extracts) ab 352.32±0.67a 369.31±26.87 386.49±17.36a 504.18±32.10b 355.71±31.35ab 620.80±4.08c 341.05±14.89b 697.36±6.15d 312.65±24.13cb 637.84±8.13cd 273.94±1.76d 673.24±5.96d 441 Note: Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%, 442 95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75% 443 EEP, 95% EEP and 100% EEP, respectively. TPC was expressed as milligram of gallic acid equivalent 444 per gram of propolis extracts (mg GAE/g extracts). TFC was expressed as milligram of rutin equivalent 445 per gram of propolis extracts (mg RE/g extracts). Dates are mean ±standard deviation (n=3).Values in 446 the same column followed by the same lower case letter are not significantly different by Duncan ’s 447 multiple range test (p<0.05). 448 21 449 450 Table 2 Inhibition of propolis extracts against yeast and rat intestinal alpha-glucosidase IC50(μg/ml) Aqueous ethanol extracts of propolis (EEP) Baker’s yeast alpha-glucosidase Rat intestinal sucrase Rat intestinal maltase WEP 16.0±1.38bc 37.91±0.52ab 32.67±0.24e 25% EEP 11.32±2.23ab 40.25±0.97ab 44.80±0.25d 50% EEP 12.90±1.14b 53.12±0.95b 100.60±2.95b 75% EEP 7.24±1.16a 32.34±0.61a 71.82±2.95c 95% EEP 16.26±0.43bc 32.91±0.54a 102.76±1.47b c ab 131.12±1.83a 100% EEP 20.01±1.54 Acarbose 177.47±6.28d 38.06±0.56 538.30±26.69c 22.46±0.86f 451 Note: Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%, 452 95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75% 453 EEP, 95% EEP and 100% EEP, respectively. Dates are mean ±standard deviation (n=3).Values in the 454 same column followed by the same lower case letter are not significantly different by Duncan’s 455 multiple range test (p<0.05). Inhibition of propolis extracts against yeast and rat intestinal 456 alpha-glucosidase were both expressed as IC50 (concentration of total phenolics able to scavenger 50% 457 of alpha-glucosidase activity) 458 459 460 461 462 22 463 464 Table 3 Inhibitory kinetics and Ki values of various propolis extracts against baker’s yeast alpha-glucosidase Aqueous ethanol extracts of propolis (EEP) WEP 25% EEP 50% EEP 75% EEP 95% EEP 100% EEP 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 Kinetic mode Ki (μg/ml) competitive inhibition competitive inhibition mixed inhibition noncompetitive inhibition mixed inhibition mixed inhibition 10.43±1.41b 9.85±2.48b 9.92±0.43b 15.18±0.58a 9.65±1.54b 10.89±2.84b Note: Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%, 95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75% EEP, 95% EEP and 100% EEP, respectively. The inhibition constant of propolis on alpha-glucosidase was expressed Ki. Dates are mean ± standard deviation (n=3).Values in the same column followed by the same lower case letter are not significantly different by Duncan’s multiple range test (p<0.05). 23 499 (A) Control 1/V(mM/min)-1 100 0.3mg/ml 80 1.0mg/ml 60 1.5mg/ml 40 20 0 -6 -3 0 3 -20 9 12 15 -40 500 501 6 1/[S](mM)-1 (B) 180 Control 0.5mg/ml 150 1/V(mM/min)-1 1.0mg/ml 120 1.5mg/ml 90 2.0mg/ml 60 30 0 -6 -4 -2 0 2 4 6 -30 1/[S ](mM) 502 503 8 10 12 14 -1 -60 (C) Control 180 0.5mg/ml 150 1.0mg/ml 1.5mg/ml 1/V(mM/min)-1 120 2.0mg/ml 90 60 30 0 -9 -6 -3 0 3 -30 504 505 6 -1 1/[S ](mM) 9 12 15 -60 (D) Control 90 0.1mg/ml 80 0.25mg/ml 70 0.5mg/ml 1/V(mM/min)-1 1.0mg/ml 60 50 40 30 20 10 0 506 -10 -5 0 1/[S](mM)-1 5 10 15 24 507 (E) 140 Control 0.5mg/ml 120 1.0mg/ml 1.5mg/ml 80 2.0mg/ml 1/V(mM/min)-1 100 60 40 20 0 -6 -4 -2 -20 2 4 6 8 10 12 14 1/[S](mM)-1 -40 508 509 0 (F) 1/V(mM/min) -1 Control 160 0.5mg/ml 140 1.0mg/ml 120 1.5mg/ml 2.0mg/ml 100 80 60 40 20 -6 -4 -2 0 -20 0 -40 510 511 512 513 514 515 2 4 6 8 10 12 14 1/[S](mM)-1 -60 Fig. 1 Lineweaver-Burk plots of inhibition kinetics of yeast alpha-glucosidase inhibitory effects by WEP (A), 25% EEP (B), 50% EEP (C), 75% EEP (D), 95% EEP (E) and 100% EEP (F). Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%, 95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75% EEP, 95% EEP and 100% EEP, respectively. 25