DEVELOPMENT AND APPLICATION OF A HIGHTHROUGHPUT SCREENING ASSAY FOR DISCOVERY OF STARCH HYDROLASE INHIBITORS LIU TING TING NATIONAL UNIVERSITY OF SINGAPORE 2013 DEVELOPMENT AND APPLICATION OF A HIGHTHROUGHPUT SCREENING ASSAY FOR DISCOVERY OF STARCH HYDROLASE INHIBITORS LIU TING TING (BSc, MSc. Wageningen University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLRATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in this thesis. This thesis has also not been submitted for any degree in any university previously. ____________________________________ Liu Ting Ting 29 July 2013 i Acknowledgments First and foremost, I would like to express my sincere gratitude to my supervisor of my doctoral study, associate professor Huang Dejian for the valuable guidance and advice. With his encouragement, I would be able to carry on my study and complete this thesis. I also would like to thank Dr. Zhang Dawei from Division of Chemistry & Biological Chemistry, Nanyang Technological University, for his kindly help on my research. Besides, I would like to thank the staff of Food Science and Technology Program of NUS, Ms. Lee Chooi Lan, Ms. Lew Huey Lee and Ms. Jiang Xiaohui for their kindly help during my study. I also would like to thank Ms. Soh Yee Lyn, Ms. Chen Mei Juan and Ms. Yew Mun Yip for their contributions in various experiments. I wish to acknowledge Jing Brand Co. Ltd. for the financial support of my research project and the Singapore International Graduate Award (SINGA) for PhD scholarship. Last but not least, I would like to extent my gratitude to my entire family for their moral support and courage throughout the years. ii Table of contents Acknowledgments ii Table of contents iii Summary vii List of tables ix List of figures . x List of abbreviations xiii Chapter 1. Introduction . - Chapter 2. Literature review . - 2.1 Starch hydrolase - 2.1.1 Starch digestion by starch hydrolase . - 2.1.2 Starch hydrolase used in diabetes research - 2.2. Methods of determining starch hydrolase activity . - 2.2.1 Conventional methods . - 2.2.2 Turbidity measurement applications - 13 2.3 Starch hydrolase inhibitors . - 14 2.3.1 Acarbose - 14 2.3.2 Phytochemicals as starch hydrolase inhibitors - 16 2.3.3 Proanthocyanidins - 16 2.3.4 Phenylpropanoid sucrose esters . - 19 2.4 Functional food for diabetes patients - 21 Chapter 3. A high-throughput assay for quantification of starch hydrolase inhibition based on turbidity measurement - 23 3.1 Introductions . - 24 3.2 Materials and methods - 27 iii 3.2.1 Reagents and instruments - 27 3.2.2 Determination of α-glucosidase activity - 29 3.2.3 Determination of dynamic range of the turbidity measurement. . - 30 3.2.4 High-throughput assay of starch hydrolase activity - 31 3.2.5 Determination of inhibitor activity - 33 3.2.6 Determination of precision and accuracy - 33 3.2.7 Statistical Analysis . - 34 3.3 Results and discussions . - 35 3.3.1 Dose response between turbidity and starch concentration . - 35 3.3.2 Quantification of the enzyme activity - 36 3.3.3 Inhibitor activity - 38 3.3.4 Precision and accuracy……………………………………………- 403.3.5 Acarbose equivalence. . - 42 3.4 Conclusion - 49 Chapter 4. Amylase and sucrase inhibition activity of diboside A isolated from wild buckwheat (Fagopyrum dibotrys) . - 50 4.1 Introduction . - 51 4.2 Materials and methods - 53 4.2.1 Assay guided fractionation of α-amylase inhibitors diboside A from Fagopyrum dibotrys - 53 4.2.2 α-Amylase inhibition assay . - 54 4.2.3 α-Glucosidase inhibition assay - 55 4.2.4 Inhibition kinetics of Diboside A - 56 4.2.5 Molecular docking study. - 57 4.2.6 Statistical analysis - 57 4.3 Results and discussions . - 58 4.3.1 Fractionation of α-Amylase inhibitors from F. dibotrys extracts - 58 4.3.2 Diboside A . - 63 4.3.3 Inhibition kinetics of diboside A on α-amylase and sucrase. - 64 4.4 Conclusion - 75 iv Chapter 5. Isolation of phenylpropanoid sucrose esters compounds from Smilax glabra rhizomes . - 76 5.1 Introduction . - 77 5.2 Material and Methods . - 79 5.2.1 Plant material and reagents - 79 5.2.2 Extractions . - 79 5.2.3 α-Amylase and α-glucosidase inhibitory study on raw extracts - 80 5.2.4 Fractionation and isolation of bioactive components from S. glabra rhizome . - 80 5.2.5 α-Amylase and α-glucosidase inhibitory study on isolated compounds81 5.3 Result and Discussion . - 83 5.3.1 α-Amylase and α-glucosidase inhibition study on raw extracts - 83 5.3.2 Structure identification of phenylpropanoid sucrose esters compounds from Smilax.glabra rhizome . - 86 5.3.3 α-Amylase and α-glucosidase inhibition activity of isolated compounds - 91 5.4 Conclusion - 96 Chapter 6. Characterization of proanthocyanidins in wild buckwheat root extracts . - 97 6.1 Introduction . - 98 6.2 Material and methods - 99 6.2.1 Extraction and characterization of proanthocyanidins - 99 6.2.2 Thiolysis of the proanthocyanidins for HPLC analysis . - 99 6.2.3 α-Amylase inhibition assay. - 100 6.2.4 α-Glucosidase inhibition assay - 101 6.3 Results and discussions……………………………………………… -1026.3.1 Characterization and quantification of proanthocyanidins isolated from Fagopyrum dibotrys rhizome by HPLC-MS. - 102 6.3.2 Thiolyzed products of the proanthocyanidin . - 107 v 6.3.3 Inhibition activity of wild buckwheat proanthocyanidins on α-amylase and α-glucosidase . - 109 6.4 Conclusions . - 111 Chapter 7. Effects of grape seeds extracts on sensory properties and in vitro digestibility of wheat noodles - 112 7.1 Introduction . - 113 7.2 Materials and methods - 115 7.2.1 Reagents and instruments - 115 7.2.2 Determination of IC50 value of grape seed extracts . - 115 7.2.3 Determination of grape seed extracts total phenolic content . - 116 7.2.4 Determination of proanthocyanidin profile of grape seed extracts-1167.2.5 Formulation of GSE incorporated wheat noodles . - 117 7.2.6 In vitro digestion model of GSE- incorporated noodle samples - 117 7.2.7 Determination of grape seeds proanthocyanidin cooking loss - 118 7.2.8 Sensory evaluation and consumer acceptance . - 119 7.3. Results and discussions - 120 7.3.1 Determination of total phenolic content of grape seeds extracts . - 120 7.3.2 Determination of the proanthocyanidin profiles of grape seeds extracts - 120 7.3.3 Determination of IC50 values of the grape seed extracts . - 123 7.3.4 Formulation of grape seed wheat noodles . - 126 7.3.5 In vitro digestion of GSE-incorporated wheat noodles . - 128 7.3.6 Determination of the loss of proanthocyanidin during cooking - 133 7.3.7 Sensory evaluation and consumer acceptance . - 135 7.4 Conclusion - 138 Chapter 8.Conclusions and future work…………………………… .- 139 List of publications . - 145 Bibliography . - 146 Appendices - 165 vi Summary Several novel approaches to treat Type diabetes have been tested, including preventing hyperglycemia by the consumption of low glycemic index (GI) foods. To search for the active ingredients in low GI foods, a 96well based high-throughput method (HTS) for rapidly measuring the inhibition of starch hydrolase was developed, by monitoring the decrease in turbidity over time during the enzymatic digestion of starch. The area under the curve of turbidity measured over time was used to quantify the inhibitory effects of polyphenolic compounds on starch hydrolase. Acarbose equivalence was introduced for the first time, which has the advantage of eliminating run-to-run variation, and allowing easier comparison of inhibitor activity. Using this assay, grape seed and cinnamon bark-extracts were found to be the most active polyphenol extracts in inhibiting starch hydrolase, and proanthocyanidins were identified as the active constituents. By applying HTS, extracts of wild buckwheat root (Fagopyrum dibotrys) exhibited strong starch hydrolase inhibitory activity. Using HTS as a guide, diboside A was isolated as a selective inhibitor of pancreatic α-amylase and sucrase, but not maltase. Analysis of the inhibition kinetics revealed that diboside A is a non-competitive inhibitor of sucrase (Ki = 72.4 µM) and uncompetitive inhibitor of α-amylase (Ki = 5.1 µM). Molecular docking studies then showed that the allosteric inhibition of α-amylase by diboside A is attributed to the number of hydrogen bonds formed and electrostatic interactions between the enzyme active site and inhibitor. To continue investigating the anti-diabetic effects of phenylpropanoid sucrose ester compounds (PSEs), smilaside A and smilaside D were isolated from ethyl acetate extracts of the Smilax glabra rhizome. Both compounds displayed negligible inhibitory effects towards porcine pancreas α-amylase, but weak and moderate inhibitory activity towards rat intestinal α-glucosidase, vii with IC50 values of 99 ± 3.8 and 56.8 ± 8.4 µM, respectively. This is the first report demonstrating inhibition of α-glucosidase by PSE compounds in vitro. Proanthocyanidins in the extracts of wild buckwheat root were also found to be major contributors to the inhibition of starch hydrolase. The components of proanthocyanidins were separated by a diol HPLC column, and their molecular weights were determined by ESI-MS. The results showed that B-type proanthocyanidin oligomers are the predominant form, with a mean degree of polymerization of 7.2. A dose-response of the proanthocyanidin fraction was used to calculate the IC50, which was 10.7 µg/mL, 35.2 µg/mL and 18.0 µg/mL for amylase, maltase, and sucrase, respectively. The use of grape seeds extracts (GSE) as α-amylase inhibitors using wheat noodles as the food matrix was investigated. Wheat noodles were formulated with the addition of 0.5, 1.0, or 2.0% GSE. A hour in vitro digestion kinetic study showed a good dose response for inhibiting the rate of starch hydrolysis. Five minutes of cooking at 100 ºC led to a 15.7% loss of proanthocyanidins and the degradation of large proanthocyanidin oligomers into smaller oligomers. Finally, a sensory test showed that the color of the noodles could significantly influence the acceptance score, and 2.0% GSE incorporation gave the highest consumer acceptance. viii Peak Epicatechin Intens. [%] -MS, 32.1min #1390 290.5 100 80 60 40 535.4 579.3 20 499.6 455.8 326.3 372.0 393.9 559.3 477.6 601.2 665.0 200 300 400 500 - 194 - 600 700 m/z Peak Epicatechin gallate Intens. [%] +MS, 34.8min #1537 212.2 100 80 234.0 60 40 443.3 180.3 20 309.7 160.5 258.0 198.2 293.8 369.6 273.9 391.7 419.7 465.5 501.3 519.2 612.2 200 250 300 350 400 - 195 - 450 500 550 600 m/z Peak4 4β-(carboxymethyl)sulphanyl-(-)-epicatechin methyl ester Intens. [%] +MS, 36.8min #1657 395.6 100 80 212.2 60 234.0 152.5 289.9 40 180.3 20 443.3 309.7 220.1 160.4 198.3 258.0 274.1 417.5 477.4 327.8 493.3 553.2 150 200 250 300 350 - 196 - 400 450 500 550 m/z Peak 4β-(carboxymethyl)sulphanyl-(-)-(epi)catechin gallate methyl ester. Intens. [%] +MS, 40.9min #1905 395.6 100 80 60 40 289.9 20 212.2 234.0 152.6 180.4 417.5 443.3 493.3 100 200 300 400 - 197 - 500 600 m/z Peak 4β-(carboxymethyl)sulphanyl-(-)-(epi)catechin gallate methyl ester. Intens. [%] +MS, 41.9min #1979 547.3 100 80 60 40 212.2 234.1 20 152.5 180.3 443.3 257.9 569.2 309.7 645.0 100 200 300 400 500 - 198 - 600 700 800 m/z Appendix 18 Grape seeds inhibitory effect on amylase - 199 - Appendix 19 Gallic acid calibration curve for total phenolic content quantification Abs at 765nm 1.6 1.2 0.8 y = 0.0598x + 0.0906 R² = 0.9938 0.4 0 10 20 Gallic acid conc. (µg/mL) - 200 - 30 Appendix 20 LC-MS/MS2 result of Grape seeds extracts determine cooking loss HPLC profiles of uncooked Tianding GSE 20mg/mL - 201 - HPLC profiles of cooked Tianding GSE 20 mg/mL - 202 - HPLC profiles of Johnson GSE - 203 - Appendix 21 Epicatechin calibration curve - 204 - Appendix 22 Sensory evaluation of grape seeds wheat noodles Sensory evaluation test Introduction: Developed product is the incorporation of herb extract into noodles as a functional food Instructions: Please rinse your mouth with water provided before starting. You can rise at any time during the test if you need to. Please taste the samples according to the number stated on each page. Do not go back and re-taste the samples once you are in another page. If you have any questions, please not hesitate to ask the server. Survey: How often you consume noodles? o More than once a week o More than once a month o More than once every months o More than once a year o Never - 205 - Questions: Indicate clicking on the box on how you feel the product rates in each category below (scale of to 9). Please click on the next attribute on the right after selection. Thank you! _______________________________________________________________ 1. Appearance – Color Too light Just right Too dark 2. Taste - Astringency Not astringent at all Just right Very strong astringent flavour 3. Texture - Firmness Too soft and soggy Just right Too firm 4. Overall opinion Like extremely Like moderately Neither like nor dislike Dislike moderately Dislike extremely Note: Astringency is the puckering sensation upon consumption End: Thank you for your participation! - 206 - Appendix 23 Glucose calibration curve of GOD/POD method application - 207 - Appendix 24 Maltose calibration curve - 208 - Appendix 25 Glucose calibration curve - 209 - [...]...List of tables Table 1 Inhibitors concentrations tested for starch hydrolases Table 2 Determination of the linear range of gelatinized starch Table 3 Accuracy and precision of the high- throughput method Table 4 IC50 and acarbose equivalence of inhibitors obtained by starch hydrolases Table 5 Estimation of the proanthocyanidins contents expressed as milligram epicatechin equivalent per 100 mg of selected... Acarbose Acarbose is a pseudo-tetrasaccharide, because of its structural similarity to typical tetrasaccharides It is bio-synthesized by Actinoplanes, a type of bacteria Acarbose strongly and broadly inhibits the brush-border enzymes glucoamylase, dextrinase, maltase, and sucrase, as well as pancreatic αamylase in vitro and in vivo [34] Due to the presence of intramolecular nitrogen (Figure 4), acarbose... than human salivary amylase (HSA) [20, 23] The role of HSA in starch digestion is relatively minor, because food is only chewed for a short time, and so salivary amylase is not the main focus of antihyperglycemia research Instead, pancreas α-amylase and α-glucosidase are the two main targets Rat intestinal acetone powder is commonly used as αglucosidase by researchers, although the extracts contain... insulin-secretagogues, insulin sensitizers, insulin-like growth factors, aldose reductase inhibitors, α-glucosidase / α-amylase inhibitors, and protein glycation inhibitors [33] The inhibition of αglucosidase and α-amylase can significantly reduce the post-prandial increase in blood glucose after a meal, with starch as the major calorie intake The apparent advantage of the starch hydrolase inhibitors is that... pancreatic α-amylase The resultant mixture of oligosaccharides is then broken down to glucose by α-glucosidases α-Amylase is an endo-acting enzyme that catalyzes the hydrolysis of α1,4-glucan bonds in starch It liberates glucose molecules in units of two or three, and the combined action of salivary and pancreatic amylase leads to the production of large amounts of maltose and maltotriose, and relatively... non-reducing end of a chain [21] In humans, sucrase-isomaltase is far more abundant than maltase-glucoamylase, and is responsible for 80% of the maltase (and maltotriase) activity in the small intestine [22] The α-(16) glucosidic bonds in starch are almost exclusively hydrolyzed by the isomaltase subunit of sucrase-isomaltase -7- Chapter 2 2.1.2 Starch hydrolase used in diabetes research α-Amylase is widely... parameter for the quantification However, it is important to consider that starch and gels are macromolecules, and the movement and structural change of macromolecules significantly affects the accuracy of measuring the transmittance at the initial stage of the reaction - 13 - Chapter 2 2.3 Starch hydrolase inhibitors Medicines for controlling diabetes and its complications can be mainly divided into... diboside A in BS1 shown as molecular surface Regions in red represent electronegative areas, regions in blue represent electropositive areas, and white areas are neutral (B) 2-D ligand interaction diagram of diboside A in BS1 Figure 19 (A) Binding conformation of diboside A in BS2 shown as molecular surface (B) 2-D ligand interaction diagram in BS2 Figure 20 Extraction, fractionation and isolation of active... extracts Table 6 IC50 and acarbose equivalence (AE) of different inhibitors on αamylase and α-glucosidase (maltase and sucrase) Table 7 Docking scores of diboside A in the 2 allosteric binding sites The cells colored in green indicate favourable interactions while cells colored in red indicate unfavorable interaction All calculated values are in kcal mol-1 Table 8 The ESI-MS/MS2 peaks of proanthocyanidins... by amylase to quantify α-amylase activity [31] The amylase activity was calculated from initial changes in absorbance caused by starch digestion Similarly, Ichiki et al., (2007) adopted this method for their amylase induction inhibition study using 96-cell microplates, a spectrophotometer, and starch- agar gel as the substrate [32] Both studies measured the initial rate as a key kinetic parameter for . introduced for the first time, which has the advantage of eliminating run-to-run variation, and allowing easier comparison of inhibitor activity. Using this assay, grape seed and cinnamon bark-extracts. exhibited strong starch hydrolase inhibitory activity. Using HTS as a guide, diboside A was isolated as a selective inhibitor of pancreatic α-amylase and sucrase, but not maltase. Analysis of the inhibition. extracts Table 6 IC 50 and acarbose equivalence (AE) of different inhibitors on α- amylase and α-glucosidase (maltase and sucrase) Table 7 Docking scores of diboside A in the 2 allosteric