INVESTIGATING THE DIGESTIVE ENZYMES INHIBITORS OF BEAN

EXTRACTS

Kiu Yan Yu Foo Toon Min

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

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)

Rationale

The large scale utilization of chemicalThe large scale utilization of chemical pesticides has caused detrimental effects pesticides has caused detrimental effects

to human health and environmentto human health and environment

Need to find new and Need to find new and natural alternative natural alternative pesticides which has pesticides which has minimal impact on human minimal impact on human health and environmenthealth and environment

Enzyme inhibitors could provide a key role inEnzyme inhibitors could provide a key role in

plant defence against pest by inhibiting major plant defence against pest by inhibiting major

insect digestive enzymes thus hampering the insect digestive enzymes thus hampering the

growth of these insects.growth of these insects.

Rationale

Enzyme inhibitors from beans, beingEnzyme inhibitors from beans, beinga type of antinutrient, havea type of antinutrient, have

been known to cause diarrhea and been known to cause diarrhea and flatulence when consumed.flatulence when consumed.

This is caused by an This is caused by an increase in resistant increase in resistant (undigested) starch passing (undigested) starch passing into the colon, where it is into the colon, where it is broken down by flatulence-broken down by flatulence-causing bacteria.causing bacteria.

There is also a need to characterize theThere is also a need to characterize the nature of these enzyme inhibitors tonature of these enzyme inhibitors to

find out how they can be deactivated.find out how they can be deactivated.

OBJECTIVES

• To determine the alpha-amylase inhibitory To determine the alpha-amylase inhibitory activity of various bean extracts. activity of various bean extracts.

• To separate and identify the fragment or To separate and identify the fragment or fragments responsible for the inhibition of fragments responsible for the inhibition of these digestive enzymes.these digestive enzymes.

• To find out the effect of different treatment To find out the effect of different treatment on these enzyme inhibitors in bean extractson these enzyme inhibitors in bean extracts

HYPOTHESIS• Different bean extracts will have varying Different bean extracts will have varying

levels of amylase inhibitory activity.levels of amylase inhibitory activity.• The bean extracts contains one or more The bean extracts contains one or more

fragments responsible for digestive fragments responsible for digestive enzyme inhibition.enzyme inhibition.

• Certain treatments, such as boiling and Certain treatments, such as boiling and soaking the beans before consumption soaking the beans before consumption would decrease its enzyme-inhibitory would decrease its enzyme-inhibitory activity.activity.

INDEPENDENT VARIABLES Types of bean usedTypes of bean usedMung Bean (Mung Bean (Vigna radiataVigna radiata))Kidney Bean (Kidney Bean (Phaseolus vulgarisPhaseolus vulgaris ) )Rice Bean (Rice Bean (Vigna umbellataVigna umbellata ) )Black-eyed Bean (Black-eyed Bean (Vigna unguiculata Vigna unguiculata

unguiculata unguiculata )) Type of treatmentType of treatment

DEPENDENT VARIABLES

• Enzyme ActivityEnzyme ActivityAmylase Inhibition Assay Amylase Inhibition Assay D Decrease

in the amount of maltose liberated in percentage

CONSTANT VARIABLES

Extraction method Extraction method Volume and Concentration of starch Volume and Concentration of starch

solutionsolution Temperature of incubationTemperature of incubation Duration of IncubationDuration of Incubation Volume and concentration of the Volume and concentration of the

enzymes, substrates and bean enzymes, substrates and bean extract usedextract used

MATERIALS

• Types of BeansTypes of Beans

Mung Bean(Vigna radiata)

Kidney Bean(Phaseolus vulgaris )

Rice Bean(Vigna umbellata )

Black-eyed Bean(Vigna unguiculata

unguiculata )

MATERIALS• 20mM sodium phosphate buffer, pH 6.9, containing 20mM sodium phosphate buffer, pH 6.9, containing

300mM NaCl300mM NaCl• 2mg/ml standard maltose solution2mg/ml standard maltose solutionAmylase Inhibition AssayAmylase Inhibition Assay• 3,5-Dinitrosalicylic acid solution3,5-Dinitrosalicylic acid solution (reacts with (reacts with

reducing sugars to form a colored compound which reducing sugars to form a colored compound which absorbs light strongly at 540 nm.absorbs light strongly at 540 nm.

• 50 mM Sodium Phosphate, 50 mM Sodium Chloride, 0.5 50 mM Sodium Phosphate, 50 mM Sodium Chloride, 0.5 mM Calcium Chloride,0.1% Bovine Serum Albumin Buffer, mM Calcium Chloride,0.1% Bovine Serum Albumin Buffer, pH 6.9pH 6.9 (or (or BSA bufferBSA buffer to stabilise the enzyme and to stabilise the enzyme and prevent adhesion of enzymes to the reaction tubes and tip prevent adhesion of enzymes to the reaction tubes and tip surfaces)surfaces)

• 1% starch solution1% starch solution• Human salivary α-Amylase solutionHuman salivary α-Amylase solution• Deionized WaterDeionized Water

APPARATUS

• Centrifudge, MicrofudgeCentrifudge, Microfudge• BlenderBlender• IncubatorIncubator• SpectrophotometerSpectrophotometer• MicropipettesMicropipettes• Hot PlateHot Plate

METHODOLOGY1. Maltose

Standard curve

2. Preparing crude Extracts of bean

3. Amylase Inhibition Assay

5. Purification andreverse HPLC

4. TreatmentTest

METHODOLOGY

Maltose standard curve• To establish the relationship between

maltose concentration and its absorbance value.

1. Maltose was used as standard solution in the concentration as 2mg/ml.

2. Pipette 1ml, 0.8ml, 0.6ml, 0.4ml and 0.2ml of maltose standard solution into respective containers

3. Make up the solution of each container to 2ml by adding different volumes of deionized water. (Blank tube contains only 2ml deionized water)

METHODOLOGY

Maltose standard curve3. Add 1ml of 1% DNS solution to each container4. Place the containers in a boiling water bath

for 5 minutes5. Cool the containers on ice to room

temperature 6. Add 8ml to each container to dilute the

solution 7. Mix the solution by inversion and record the

absorbance value for the blank and test using the spectrometer at 540nm

METHODOLOGY

Preparing Crude Extracts of Beans

1. 25g of beans were blended and homogenized in 50ml of 20mM sodium phosphate buffer , pH 6.9, containing , pH 6.9, containing 300mM NaCl300mM NaCl

2. The crude extract was then centrifuged at 8000rpm for 10 minutes.

3. Collect the supernatant in eppendorf tubes, and centrifuge again at 12,000 rpm for 20minutes.

METHODOLOGY

Amylase Inhibition Assay 1. The following reagents were added:

2. The reaction mixtures were incubated for 15 minutes at 25°C.

Blank Control solution

Bean-only solution

Bean + amylase solution

BAS Buffer (ml)

1 1 1 1

Alpha-amylase (µl)

- 10 - 10

Bean extract (µl)

- - 50 50

Deionised water (µl)

60 50 10 10

METHODOLOGY

Amylase Inhibition Assay3. 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

4. The reaction was stopped by adding 500µl DNS solution heating the contents in a boiling water bath for 15 minutes

METHODOLOGY

Amylase Inhibition Assay5. 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.

METHODOLOGY

Treatment Test

• Three types of treatment are carried out

1. Pre-soaking2. Boiling3. Dehulling (future work)

METHODOLOGY

Treatment Test1. Pre-soaking Beans were soaked in water (1:5

(weight/volume) bean to water ratio) overnight

2. Boiling Beans were heated in boiling water bath

for 30 minutes3. Dehulling Beans were manually dehulled with

sharp knife.

RESULTS AND DISCUSSION

Maltose Standard Curve• From our experiment, we have obtained

the following results and plotted the maltose standard curve:

Graph of Absorbance at 540nm against Miligrams of Maltose

R2 = 0.9999

00.10.20.30.40.50.60.70.8

0 0.5 1 1.5 2 2.5

Miligrams of Maltose

Ab

so

rba

nc

e a

t 5

40

nm

Absorbance at 540nm

Linear (Absorbance at540nm)

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.

RESULTS AND DISCUSSION

Amylase Inhibition Assay

Sample Result LabelUninhibited amylolytic

activity (amylase only)

ΔA540nm Control

a

Endogenous amylolytic

activity of bean (bean only)

ΔA540nm Bean-only

b

Inhibited amylolytic

activity (Bean + amylase)

ΔA540nm Test c

RESULTS AND DISCUSSION

Amylase Inhibition Assay• Inhibition (%) can be calculated by

the formula:

((a-(c-b) )/a)*100%

Inhibited amylolytic activity without the influence of the endogenous amylolytic activity of the bean

Decrease in absorbance value (due to decrease in amount of maltose liberated)

RESULTS AND DISCUSSION

Amylase Inhibition AssayAlpha-amylase Inhibition

Mung Bean-3.15%

Rice Bean -0.62%

Kidney Bean 84.3%

Black-eyed bean 2.99%

-20

0

20

40

60

80

100

Inhibition (%)

Inh

ibit

ion

(%

)

Mung Bean Rice Bean Kidney Bean Black-eyed bean

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.

Alpha-amylase inhibition of Kidney Bean after treatment

Untreated 84.3%

Soaked, 30.7%

Boiled 4.08%

0

20

40

60

80

100

Inhibition (%)

Inh

ibit

ion

(%

)

Untreated

Soaked

Boiled

RESULTS AND DISCUSSION

Treatment test

63.6%

95.1%

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

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.

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

APPLICATIONS

• Bean extracts with effective Bean extracts with effective digestive enzyme inhibitors can be digestive enzyme inhibitors can be further studied to make insecticides, further studied to make insecticides, increasing production yield.increasing production yield.

• Bean extracts with amylase Bean extracts with amylase inhibitory activity can also be used inhibitory activity can also be used as a novel medicine to combat as a novel medicine to combat diabetes diabetes

FUTURE WORK

• Effect of dehulling on the alpha-Effect of dehulling on the alpha-amylase inhibitory activity of bean amylase inhibitory activity of bean extractextract

• Identification and purification of Identification and purification of fragment or fragments responsible fragment or fragments responsible for the inhibition of these digestive for the inhibition of these digestive enzymes using HPLCenzymes using HPLC

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• 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(Pisum sativum ). 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.