INVESTIGATING THE DIGESTIVE ENZYMES INHIBITORS OF BEAN EXTRACTS

1. INVESTIGATING THE DIGESTIVE ENZYMES INHIBITORS OF BEAN EXTRACTS Kiu Yan Yu Foo Toon Min

2. INTRODUCTION Beans are known to be a rich source of defensins, small, cysteine rich proteins found in most plants. Some of these defensins are known to have enzyme-inhibiting properties

3. INTRODUCTION • For example, defensins from soybeans are known to inhibit human and bovine trypsin. • Defensins from the common bean has also been found to inhibit the alpha-amylase activities of adzuki bean weevil (C.chinensis L.) and cowpea weevil (C. maculatus)

4. Rationale The large scale utilization of chemical pesticides has caused detrimental effects to human health and environment Enzyme inhibitors could provide a key role in plant defence against pest by inhibiting major insect digestive enzymes thus hampering the growth of these insects. Need to find new and natural alternative pesticides which has minimal impact on human health and environment

5. Enzyme inhibitors from beans, being a type of antinutrient, have been known to cause diarrhea and flatulence when consumed. Rationale There is also a need to characterize the nature of these enzyme inhibitors to find out how they can be deactivated. This is caused by an increase in resistant (undigested) starch passing into the colon, where it is broken down by flatulence-causing bacteria.

6. OBJECTIVES To determine the alpha-amylase inhibitory activity of various bean extracts. To separate and identify the fragment or fragments responsible for the inhibition of these digestive enzymes. To find out the effect of different treatment on these enzyme inhibitors in bean extracts

7. HYPOTHESIS Different bean extracts will have varying levels of amylase inhibitory activity. The bean extracts contains one or more fragments responsible for digestive enzyme inhibition. Certain treatments, such as boiling and soaking the beans before consumption would decrease its enzyme-inhibitory activity.

8. INDEPENDENT VARIABLES • Types of bean used • Mung Bean (Vigna radiata) • Kidney Bean (Phaseolus vulgaris ) • Rice Bean (Vigna umbellata ) • Black-eyed Bean (Vigna unguiculata unguiculata ) • Type of treatment

9. DEPENDENT VARIABLES • Enzyme Activity • Amylase Inhibition Assay  Decrease in the amount of maltose liberated in percentage

10. CONSTANT VARIABLES • Extraction method • Volume and Concentration of starch solution • Temperature of incubation • Duration of Incubation • Volume and concentration of the enzymes, substrates and bean extract used

11. MATERIALS • Types of Beans Black-eyed Bean (Vigna unguiculata unguiculata) Mung Bean (Vigna radiata) Kidney Bean (Phaseolus vulgaris ) Rice Bean (Vigna umbellata )

12. MATERIALS • 20mM sodium phosphate buffer, pH 6.9, containing 300mM NaCl • 2mg/ml standard maltose solution Amylase Inhibition Assay • 3,5-Dinitrosalicylic acid solution (reacts with reducing sugars to form a colored compound which absorbs light strongly at 540 nm. • 50 mM Sodium Phosphate, 50 mM Sodium Chloride, 0.5 mM Calcium Chloride,0.1% Bovine Serum Albumin Buffer, pH 6.9 (or BSA buffer to stabilise the enzyme and prevent adhesion of enzymes to the reaction tubes and tip surfaces) • 1% starch solution • Human salivary α-Amylase solution • Deionized Water

13. APPARATUS • Centrifudge, Microfudge • Blender • Incubator • Spectrophotometer • Micropipettes • Hot Plate

14. METHODOLOGY 4. Treatment Test 1. Maltose Standard curve 2. Preparing crude Extracts of bean 3. Amylase Inhibition Assay 5. Purification and reverse HPLC

15. METHODOLOGY Maltose standard curve • To establish the relationship between maltose concentration and its absorbance value. • Maltose was used as standard solution in the concentration as 2mg/ml. • Pipette 1ml, 0.8ml, 0.6ml, 0.4ml and 0.2ml of maltose standard solution into respective containers • Make up the solution of each container to 2ml by adding different volumes of deionized water. (Blank tube contains only 2ml deionized water)

16. METHODOLOGY Maltose standard curve • Add 1ml of 1% DNS solution to each container • Place the containers in a boiling water bath for 5 minutes • Cool the containers on ice to room temperature • Add 8ml to each container to dilute the solution • Mix the solution by inversion and record the absorbance value for the blank and test using the spectrometer at 540nm

17. METHODOLOGY Preparing Crude Extracts of Beans • 25g of beans were blended and homogenized in 50ml of 20mM sodium phosphate buffer , pH 6.9, containing 300mM NaCl • The crude extract was then centrifuged at 8000rpm for 10 minutes. • Collect the supernatant in eppendorf tubes, and centrifuge again at 12,000 rpm for 20minutes.

18. METHODOLOGY Amylase Inhibition Assay • The following reagents were added: • The reaction mixtures were incubated for 15 minutes at 25°C.

19. METHODOLOGY Amylase Inhibition Assay • 250µl of reaction mixture was added to 500µl of starch solution and 250 µl of deionized water before further incubating at 37°C for 10 minutes • The reaction was stopped by adding 500µl DNS solution heating the contents in a boiling water bath for 15 minutes

20. METHODOLOGY Amylase Inhibition Assay 5. The solution was allowed to cool before the volume in each tube was made up to 5 ml by adding de-ionized water and mixing the contents by inversion 6. The absorbance values of the blank, control, bean-only and test solutions were measured at 540 nm. 7. Triplicates are done for each bean extract used.

21. METHODOLOGY Treatment Test • Three types of treatment are carried out • Pre-soaking • Boiling • Dehulling (future work)

22. METHODOLOGY Treatment Test • Pre-soaking • Beans were soaked in water (1:5 (weight/volume) bean to water ratio) overnight • Boiling • Beans were heated in boiling water bath for 30 minutes • Dehulling • Beans were manually dehulled with sharp knife.

23. RESULTS AND DISCUSSION Maltose Standard Curve • From our experiment, we have obtained the following results and plotted the maltose standard curve:

24. RESULTS AND DISCUSSION Maltose Standard Curve • This shows that amount of maltose liberated is directly proportional to absorbance value. • Amylase inhibitory activity can be expressed as the decrease in the amount of maltose liberated in percentage. • Using this standard curve, we can determine the presence of amylase inhibitors denoted by a lower absorbance value for the solution containing the bean extract and amylase.

25. RESULTS AND DISCUSSION Amylase Inhibition Assay

26. RESULTS AND DISCUSSION Amylase Inhibition Assay • Inhibition (%) can be calculated by the formula: ((a-(c-b) )/a)*100% Decrease in absorbance value (due to decrease in amount of maltose liberated) Inhibited amylolytic activity without the influence of the endogenous amylolytic activity of the bean

27. Amylase Inhibition Assay RESULTS AND DISCUSSION

28. RESULTS AND DISCUSSION Amylase Inhibition Assay • From the graph, kidney bean is the only extract that has consistently inhibited human alpha-amylase. • Human alpha-amylase is also highly inhibited by it (84.3%) • Conversely, the other bean extracts recorded an average of near zero or even negative inhibition. • However in general, this shows that all but kidney bean extract did not show any significant inhibition of human alpha-amylase.

29. RESULTS AND DISCUSSION Treatment test 63.6% 95.1%

30. RESULTS AND DISCUSSION • Only the kidney bean was treated as it has the highest alpha-amylase inhibitory activity. • From the graph, soaking the bean reduced its inhibitory activity by more than 60% • Boiling the bean almost completely stopped its inhibitory activity • This shows that the inhibitor is probably a heat-labile protein

31. RESULTS AND DISCUSSION • This also suggests that presoaking and cooking the beans would have already removed most or all of the inhibitors • Therefore improving the digestibility of the beans and reducing flatulence.

32. LIMITATIONS • Absorbance values may also vary due to errors in pipetting. • Different varieties of the same species of bean may contain varying contents of enzymes inhibitors

33. APPLICATIONS • Bean extracts with effective digestive enzyme inhibitors can be further studied to make insecticides, increasing production yield. • Bean extracts with amylase inhibitory activity can also be used as a novel medicine to combat diabetes

34. FUTURE WORK • Effect of dehulling on the alpha-amylase inhibitory activity of bean extract • Identification and purification of fragment or fragments responsible for the inhibition of these digestive enzymes using HPLC

35. BIBLIOGRAPHY • Sivakumar, S. , Mohan, Franco , O.L. , Thayumanavan, B. (2006) Inhibition of insect pest-amylases by little and Wnger millet inhibitors. Pesticide Biochemistry and Physiology ,85, 155–160. • Kokiladevi, E., Manickam, M. , Thayumanavan, B. (2005) Characterization of Alpha-amylase inhibitor in Vigna sublobata. Botanical Bulletin of Academia Sinica, 46, 189-196 • Broekaert, W. F., Cammue, B. P., De Bolle, M. F., Thevissen, K., De Samblanx, G. W., & Osborn, R. W. (1997). Antimicrobial Peptides from Plants. Critical reviews in plant sciences (16), 297-323. • Broekaert, W. F., Terras, F. R., Cammue, B. P., & Osborn, R. W. (1995). Plant Defensins: Novel Antimicrobial Peptides as Components of the Host Defense System. Plant Physiol (108), 1353-1358. • Hartmut, E. S., Stephanie ,G., Andrew, M., Linda, M.T., Stuart, C., & Darryl,C. H.,(1995)Bean a-Amylase lnhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas(Pisumsativum ). Plant Physiol (107), 1233-1239 • Osborn, R. W., Samblanx, G. W., Thevissen, K., Goderis, I., Torrekens, S., & Leuven, F. V. (1995). Isolation and characterisation of plant defensins from seeds of Asteraceae,. FEBS Letters (368), 257-262.

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