In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf ·...

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In Vitro Antioxidant and α-Amylase Inhibition Activities of Spiced Red Chili Paste (Datta) from South Ethiopia Conference place : Desalegn Hotel By: Dr. Engida Dessalegn ([email protected] )

Transcript of In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf ·...

Page 1: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

In Vitro Antioxidant and α-Amylase Inhibition

Activities of Spiced Red Chili Paste (Datta) from

South Ethiopia

Conference place : Desalegn HotelBy: Dr. Engida Dessalegn ([email protected])

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Content

• Introduction

• Material and Methods

• Total phenolic (TPC) and total flavonoid (TFC)

• Antioxidant Activity

- DPPH scavenging

- Ferric reducing power

- Total antioxidant activity

• In vitro alpha-amylase inhibition activity (DNSA method)

• Results

-TPC/TFC

-Antioxidant activity (DPPH, ferric reducing, phosphomolybdenum)

-Alpha-amylase inhibition activity (DNSA method)

• Correlation analysis

• Conclusion

• Recommendations

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Introduction

• Spices & herbs are most ancient and traditional products in Ethiopia.

• Ethiopia is among the largest consumer in Africa

• The major uses:

preparation of spiced ground red pepper (Berbere).

preparation of spiced chili paste (Datta).

flavor milk, bread, butter, meat, soups, and different vegetables.

as medicines.

• Rich sources of secondary metabolites (large family of phytochemicals).

Page 4: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Introduction…

• Spiced chili paste (Datta) is a traditional food in the southern part of Ethiopia, consumed mainly with raw meat and known to improve appetite.

• In general, the ingredients used in the paste are ginger, garlic, basil (leaf), korarima (seed), coriander (leaf and fruit) and chili pepper (red or green).

• The ratio and types of spices and herbs used may differ from home to home or region to region (Siripongvutikorn et al., 2008) that may also affect antioxidant activity.

• The product is commonly available in local market and

supermarkets.

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Introduction…

• What are free radicals?

• Oxidants (ROS & RNS)

• Highly reactive compounds created in the body or introduced from the environment. (radiation, smoking etc)

• Oxidation and damage of "cellular molecules" such as proteins, lipids and DNA.

• If not eliminated, may lead to chronic diseases: Alzheimer’s, cancer, atherosclerosis, diabetes mellitus, hypertension and aging.

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Introduction…

• What are antioxidants?

• A substance that inhibits oxidation or reactions

promoted by free radicals.

• Protect organs from excess ROS/RNS ( protecting

from oxidative damage).

• Synthetic and natural antioxidants

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Introduction…

• Synthetic antioxidantbutylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)

are used as food additives.

associated with liver damage and carcinogenesis.

• Natural antioxidants

• Sources: vegetables, fruits, spices, herbs, cereals etc.)

• Why natural antioxidants?

• variety of structures and chemical interactions.

• numerous biological activities they can perform.

(antimicrobial, anticancer, antidiabetic, etc)

• Save

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Introduction…

• Phytochemicals are Bioactive, naturally occurring plant products.

• Phenolic compounds, large family of phytochemicals(flavonoids, phenolic acids, tannins etc.).

• Present in foods of plant origin and medicinal plants.

• Diets rich in phenolic compounds have many health benefits in relation to antioxidant activity: antimicrobial, antidiabetic, anticancer etc.

• Phenolic compounds scavenge free radicals originating from different oxygen and nitrogen species (ROS/RNS).

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Introduction…

• superoxide anion (O2•−), hydroxyl radical (HO•) peroxyl

radical (ROO•), nitric oxide (NO•), hydrogen peroxide

(H2O2), and peroxynitrite (ONOO•−).

OH

+ ROO+ RCOOH

O

Phenol Free radiclal Phenoxide radical

Page 10: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Introduction…

Type 2 diabetes mellitus is the most encountered form of diabetes, accounting for more than 80% of the total cases of diabetes.

Prolonged exposure to elevated glucose induces the production of free radicals, particularly reactive oxygen species (ROS), through glucose auto-oxidation and protein glycosylation.

The non-enzymatic glycation of proteins (Maillardreaction) is a process closely linked to oxidative stress and is associated with the formation of complex compounds (AGE).

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Introduction…

Both synthetic compounds and natural products have been evaluated as inhibitors against the formation of AGEs.

Many synthetic inhibitors were withdrawn from clinical trials due to relatively low efficacies and unsatisfactory safety .

most people have limited resources and do not have access to modern treatment.

Plants have long been used for the treatment of diabetes, particularly in developing countries.

α-Amylase

– Catalyzes digestion of starch and oligosaccharides

– Inhibitors of digestive enzymes slow the absorption of carbohydrates.

– Phenolic compounds inhibit the activity of alpha-amylase

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Objectives

• Determine the TPC and TFC contents.

• Evaluate in vitro antioxidant activity.

• Determine the in vitro alpha-amylase inhibition activity

• Evaluate the relationship between TPC/TFC and antioxidant and alpha amylase inhibition activity to establish phenolic constituents contributions.

Page 13: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology

• Sample collection & preparation of Datta paste

Coriander (fruit)

Datta paste

Chili pepperGinger

Garlic

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Methodology…

• Purchased from local market in Hawassa town, South Ethiopia, in October, 2012.

• All samples sorted and washed thoroughly to remove dust and dirt.

• 100 g of chili pepper and 10 g from each spice and herbs were pounded together and then the resulting paste was salted.

• Paste was freeze-dried (Model 2085C0000, Kinetics Thermal Systems, Stone Ridge, NY, USA) and then ground to fine powder using electric grinder (FM100 model, China).

• Sample was stored at −20 oC until used for the in vitro assays.

Page 15: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology…

• Extraction of Datta paste• Freeze dried sample soaked in solvents- pet.

ether, water, acetone, methanol, and methanol (80%) for 6 h at room temperature, in a mechanical shaker.

• Filtered and evaporated to

dryness with a rotary evapor.

• stored in a sealed plastic

bottles at -20 oC

• . Fig 1 Freeze drier

Page 16: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology…

• TPC

• Folin-ciocalteu method

• The absorbance of the

resulting blue color was

measured at 765 nm

with a UV- visible

spectrophotometer

• Expressed as mg GAE/g

of dried extract.

0 25 50 75 100 125

0.0

0.5

1.0

1.5

Ab

so

rba

nc

e (

76

5 n

m)

Concentration (g/mL)

y = 0.02x + 0.09, R2 = 0.99

A

Fig 2. Gallic acid calibration curve

Page 17: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

TFC

• Flavonoid-aluminum complex

• Absorbance at

415 nm were taken after 1 h of

incubation

• TFC Expressed as

mg QE/g of dried extract 0 10 20 30 40

0.0

0.3

0.6

0.9

1.2

Ab

so

rba

nc

e (

41

5 n

m)

Concentration (g/mL)

y = 0.024x + 0.11, r2 = 0.98

B

Fig 3 Quercetin calibration curve

Page 18: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

• Antioxidant activity1. DPPH scavenging• 1mL of the extract (10-1000µg/ml)

• DPPH radical dissolved in methanol

• The absorbance was taken at

• 520 nm using a spectrophotometer.

• DPPH scavenging (%) = [(A0 -At)/A0)] × 100

• Ao –absorbance of control, At- absorbance of sample

• BHT and ascorbic acid were used as a reference standard.

Page 19: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

2. Reducing Power Activity

(Iron (III) to iron (II) reduction)

The concentration of Fe 2+ is monitored by

measuring the formation of Perl’s Prussian blue.

0 25 50 75 100 125

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abso

rban

ce (7

00 n

m)

Concentration (g/mL)

y = 0.022x + 0.26, R2 = 0.98

A

Fig 4 Ascorbic acid calibration curve

Fe3+

Fe2+

extract

yellow blue

Page 20: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

3. Phosphomolybdenum assay

• Based on the reduction of Mo (VI) to Mo (V)

formation of green Mo (V) complexes in acidic

medium.

• Plant extract was mixed with the reagent solution (0.6

M sulphuric acid, 28 mM sodium phosphate and 4 mM

ammonium molybdate).

• The samples were incubated at 95 oC for 90 min,

cooled to room temperature and absorbance was

measured at 695 nm and methanol was used as blank.

Page 21: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

• The antioxidant activity was expressed as milligram butylated

hydroxytoluene equivalent/gram of dried extract (mg BHTE/g)

based on the calibration curve.

0.0 0.4 0.8 1.2

0.0

0.2

0.4

0.6

Abs

orba

nce

(695

nm

)

Concetration (mg/mL)

y = 0.43x + 0.08

R2

= 0.99 (p < 0.01)

B

Fig 5 BHT calibration curve

V 6+

V5+

extract

Colorless Green

Page 22: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

Alpha-amylase inhibition activity• 1 mL of α-amylase (from Aspergillus oryzae) was added to test

samples 1 mL (1 mg/mL) in phosphate buffer solution.

• 1 mL of 1% boiled potato starch solution in phosphate buffer solution was added.

• After incubation, the reaction was stopped by adding 1 mL of DNSA color reagent (1.0 g of 3, 5- dinitrosalicyclic acid, 20 mL of 2 M NaOH and 30 g of sodium potassium tartarate in 100 mLdistilled water).

• The sample test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature.

• Absorbance was read at 540 nm

Page 23: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

• Alpha-amylase inhibition activity

Starchalpha-amylase

Reducing sugar

DNSA

ANSA (brouwn, high

absorbance)

Starchalpha-amylase

Reducing sugar

DNSA

ANSA (brouwn, low

absorbance)Extract Yellow

Yellow

Acarbose was used as reference.

% inhibition = [Acontrol - (Asample -Ablank)/Acontrol]x100

Page 24: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

COOH

OH

NO2O2N

CHO

OH

HO

OH

CH2OH

OH

COOH

OH

NH2O2N

3.5-Dinitrosalicylic acid

(yellow)

Glucose

3.Amino-5-nitrosalicylic

acid (red brown)

+

Gluconic acid

COOH

OH

HO

OH

OH

CH2OH

+

•Reducing sugar reduces DNSA to 3- amino-5-nitrosalicylic acid (red –brown

color).

•The magnitude of the absorbance (red –brown) is directly proportional to the

amount of reducing sugar produced as a result of hydrolysis of starch.

Fig 6 Reaction of DNSA with reducing sugar

Page 25: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Methodology...

Statistical analysis

• A triplicate data were analyzed by one way analysis

of variance (ANOVA) using SPSS 20.0 statistical

software.

• Mean separation was conducted using Duncan’s

multiple range tests at p < 0.05.

• The (IC50) was calculated from the dose–response

curves (Origin 8 software).

Page 26: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result and Discussion

TPC/TFC

Extract Total phenolic

(mg GAE/g)

Total flavonoid

(mg QE/g)

Petroleum ether 9.69 ± 0.87b 8.82 ± 0.58d

Water 7.22 ± 1.21a -

Acetone 14.98 ± 0.76d 22.05 ± 0.87c

Methanol 8.55 ± 0.73ab 5.17 ± 0.08a

Aqueous: methanol

(20:80, v/v)

11.64 ± 0.43c 16.19 ± 0.14b

Table 1 TPC (mg GAE/ g DE) andTFC (mg QE/g DE) of various extracts of red data paste.

Page 27: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result …

1. DPPH scavenging

Plant extract

N O 2

N O 2

N O 2

N N (C 6H 5)2

D P P H

+ A H

N O 2

N O 2

N O 2

N N (C 6H 5)2

H

+ A

a n tio x id a n t D P P H -H

Page 28: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

• DPPH scavenging activity

0

25

50

75

100

0 50 100 250 500 1000

DP

PH

sca

ven

gin

g (%

)

Concentration (µg/mL)

Water

Methanol

Petroleum ether

Aqueous:methanol (20:80, v/v)

Acetone

BHT

AA

Figure7 DPPH radical scavenging activity (%) of various solvent extracts from dried Datta

paste and controls (L-ascorbic acid and BHT). Values are average of triplicate measurements

(mean ± SD).

Page 29: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

• DPPH scavenging IC50 (g/mL)

b

d

ab

c

b

ab a

0

100

200

300

400

500

600

700

800

900

1000

Petroleum ether Water Acetone Methanol Aqueous:methanol (20:80,

v/v)

BHT Ascorbic acid

IC5

0 (

µg/

mL)

23.38 g/mL36.56 g/mL

99.44 g/mL

392.13 g/mL

87.84 g/mL

795.96 g/mL

94.22 g/mL

Page 30: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

2. Ferric reducing activity

c

a

d

a

b

0

2

4

6

8

10

12

14

Petroleum ether Water Acetone Methanol Aqueous: methanol(20:80, v/v)

Ferr

ic r

ed

uci

ng

po

we

r (m

g A

AE/

g D

E)

Acetone (11.06) > pet. ether (3.10) >

methanol 80% (2.10) > methanol

(0.90) > water (0.40) mg AAE/g DE

Page 31: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

3. Phosphomolybdenum assay

b

a

c

a

b

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Petroleum ether Water Acetone Methanol Aqueous: methanol(20:80, v/v)

Tota

l an

tio

xid

ant

acti

vity

(m

g B

HTE

/g D

E)

Acetone (0.62) > pet. ether (0.43) > methanol

80% (0.32) > methanol (0.19) > water (0.18)

mg BHTE/g DE

Page 32: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

Alpha-amylase inhibition activity

C- Control, 1-Water extract (red), 3-Methanol extract (red), 5- Methanol 80% extract (red)7- Pet. ether extract (red), 9- Acetone extract (red),

Page 33: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

Alpha-amylase inhibition activity

c

a

d

c c

b

e

0

10

20

30

40

50

60

70

80

90

Petroleum ether Water Acetone Methanol Aqueous:methanol (20:80,

v/v)

Catechin Acarbose

α-a

myl

ase

inh

ibit

ion

act

ivit

y (%

)

Acarbose (74.50%) > Acetone (56.02%) >

methanol (45.60%) > pet. ether (44.95%) >

methanol 80% (44.80%) > catechin (34.51%) >

water (25.41%)

Page 34: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

0 2 4 6 8 10 12 14

0

25

50

75

A

DP

PH

scaven

gin

g (

%)

TPC (mg GAE/g of dried extract)

y = 2.7x + 34.01, r = 0.93 (p < 0.01)

0 2 4 6 8 10 12 14

0

2

4

6

8

10

12B

Fer

ric

red

uci

ng

po

wer

(m

g A

AE

/g o

f d

ried

ext

ract

)

TPC (mg GAE/g of dried extract)

y = 1.35x - 10.43, r = 0.89 (p < 0.05)

Correlation analysis

Figure 8 Correlations between TPC: DPPH scavenging (%) (A) and ferric reducing power (B)

Page 35: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

0 2 4 6 8 10 12 14

0.0

0.2

0.4

0.6

C

To

tal a

nti

oxi

dan

t ac

tivi

ty (

mg

BH

TE

/g o

f d

ried

ext

ract

)

TPC (mg GAE/g of dried extract)

y = 0.06x - 0.24, r = 0.88 (p < 0.05)

0 2 4 6 8 10 12 14

0

10

20

30

40

50

D

Alp

ha-a

myla

se in

hib

itio

n a

cti

vit

y (

%)

TPC (mg GAE/g of dried extract)

y = 3.02 + 13.26, r = 0.78 (p > 0.05)

Figure 9 Correlations between TPC: TA (C) and alpha-amylase inhibition activity (D)

Page 36: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

0 5 10 15 20 25

0

25

50

75

A

DP

PH

scaven

gin

g (

%)

TFC (mg QE/g of dried extract)

y = 0.90x + 52.53, r = 0.94 (p < 0.01)

0 5 10 15 20 25

0

2

4

6

8

10

12B

Ferr

ic r

ed

ucin

g p

ow

er

(mg

AA

E/g

of

dri

ed

extr

act)

TFC (mg QE/g of dried extract)

y = 0.41x + 0.75, r = 0.83, (p < 0.05)

Figure 10 Correlations between TFC: DPPH scavenging (%) (A) and ferric reducing power (B)

Page 37: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Result…

0 5 10 15 20 25

0.0

0.2

0.4

0.6

C

To

tal an

tio

xid

an

t acti

vit

y (

mg

BH

TE

/g o

f d

ried

extr

act)

TFC (mg QE/g of dried extract)

y = 0.018x + 0.16, r = 0.85 (p < 0.05)

0 5 10 15 20 25

0

10

20

30

40

50

D

Alp

ha-a

myla

se in

hib

itio

n a

cti

vit

y (

%)

TFC (mg QE/g of dried extract)

y = 1.03x + 33.71, r = 0.81 (p > 0.05)

Figure 11 Correlations between TFC: TA (C) and alpha-amylase inhibition activity (D)

Page 38: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Conclusion

• TPC and TFC of Datta paste were highest in the acetone extract.

• This extract showed highest value of antioxidant activity and and α-amylase inhibition activity.

• This suggests that the antioxidant and α-amylase inhibition activities of the tested extracts were closely associated with their phenolic constituents.

• Datta paste has significant antioxidant and α-amylase inhibition activities, which can be used as easily accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical applications.

Page 39: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

• Evaluation of antioxidant activity of each raw material to see the existence of synergism.

• Isolation of individual compounds to elucidate their different antioxidant mechanisms.

• Effect of the extracts on protein digestibility and sensory quality of food products in the view of the phenolic content and protein precipitation capacity of the extracts.

• Further study on toxicity

• Effect of storage on antioxidant activity and the stability of bioactive compounds on export Ethiopian spices and herbs (ginger, korarima, coriander etc).

Recommendation

Page 40: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Recommendation…

• Effect of thermal treatment (cooking temp, and time)

• further studies on the bioavailability and in-vivo

antioxidant of various solvent extracts and individual

compounds in various animal models.

• Conducting in vivo and clinical investigation on

isolated bioactive molecules for confirming the

activity of antidiabetic agents.

Page 41: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Acknowledgement

• Hawassa College of Education.

• Department of Environmental Sciences, Dalhousie Agricultural College, Canada.

Page 42: In Vitro Antioxidant and α-Amylase Inhibition Activities .... Engida Dessalegn.pdf · Alpha-amylase inhibition activity • 1 mL of α-amylase (from Aspergillus oryzae) was added

Thank you