Lesson 4.4 Total Flavonoid Content and Antioxidant Assay

Aluminum Chloride Colorimetric Technique

test used for flavonoids estimation/total flavonoid content

C4-Keto group and C-3/C-5 hydroxyl groups of flavones and flavonols

PRINCIPLE OF AlCl3 COLORIMETRIC TECHNIQUE:

a. AlCl3 forms acid stable complexes with the ________________________

b. It also forms acid labile complexes with the ________________________

a = ?

ortho-dihydroxyl groups in the A- or B-ring of flavonoids

PRINCIPLE OF AlCl3 COLORIMETRIC TECHNIQUE:

a. AlCl3 forms acid stable complexes with the ________________________

b. It also forms acid labile complexes with the ________________________

b = ?

intensity

The amount of flavonoids is based on the _______________ of the color.

intensity of the yellow solution

TOTAL FLAVONOID CONTENT:

a. Positive Result is based on the _________________________

b. It will be quantified ___________________________________

c. ___________ is used as the standard reagent for the calibration curve

d. _______________________________ is used to estimate the quantity of the flavonoids in the extract and expressed as quercetin equivalent.

a = ?

spectrophotometrically at 420 nm

TOTAL FLAVONOID CONTENT:

a. Positive Result is based on the _________________________

b. It will be quantified ___________________________________

c. ___________ is used as the standard reagent for the calibration curve

d. _______________________________ is used to estimate the quantity of the flavonoids in the extract and expressed as quercetin equivalent.

b = ?

Quercetin

TOTAL FLAVONOID CONTENT:

a. Positive Result is based on the _________________________

b. It will be quantified ___________________________________

c. ___________ is used as the standard reagent for the calibration curve

d. _______________________________ is used to estimate the quantity of the flavonoids in the extract and expressed as quercetin equivalent.

c = ?

Linear Regression Technique (y=mx+b)

TOTAL FLAVONOID CONTENT:

a. Positive Result is based on the _________________________

b. It will be quantified ___________________________________

c. ___________ is used as the standard reagent for the calibration curve

d. _______________________________ is used to estimate the quantity of the flavonoids in the extract and expressed as quercetin equivalent.

d = ?

prepared quercetin solution, plant extracts, and blank (methanol)

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

1 = ?

2% aluminum chloride

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

2 = ?

30 mins in a dark environment

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

3 = ?

1M NaOH

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

4 = ?

distilled water

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

5 = ?

96-well microplate

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

7 = ?

standard calibration curve and amount of quercetin equivalent of the plant extract

MAIN ASSAY:

1. In a microcentrifuge tube, add 500 mcL of the _____________________.

2. Add 500 mcL of _____________________.

3. Cover the microplate and incubate the mixture for ___________________.

4. After incubation, add 250 mcL of _________________.

5. Dilute the mixture with 250 mcL of _________________.

6. Transfer an aliquot of 200 mcL of the reaction mixture in a ____________ (perform in triplicates)

7. Determine the absorbance at ____________

8. Compute for the __________________________________

8 = ?

Radical Scavenging Activity Assay

other name for Antioxidant Assay

Reactive Oxygen Species

attack biological macromolecules in vivo, which will rise to protein, lipid, and DNA damage, cell aging, and oxidative stress-originated diseases

hydroxyl (OH), peroxyl (ROO), alkoxy radicals (RO) and hydrogen peroxide (H2O2)

examples of ROS

Antioxidants

any substance that when present at relatively low concentrations, compared witj those of the oxidizable substrate, significantly delays or inhibits oxidation of substrate

Free Radicals

ROS are also known as _______________ due to its one unpaired electron

reduction

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• based on the a.________ of DPPH in the presence of a hydrogen donating antioxidant.

• extracts reduces the b.______ of DPPH due to the power of hydrogen donating ability

• DPPH is one of the compounds that possess a c.___________________ with a characteristic absorption, which decreases significantly on exposure to proton radical scavengers

a = ?

purple color

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• based on the a.________ of DPPH in the presence of a hydrogen donating antioxidant.

• extracts reduces the b.______ of DPPH due to the power of hydrogen donating ability

• DPPH is one of the compounds that possess a c.___________________ with a characteristic absorption, which decreases significantly on exposure to proton radical scavengers

b = ?

proton free radical

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• based on the a.________ of DPPH in the presence of a hydrogen donating antioxidant.

• extracts reduces the b.______ of DPPH due to the power of hydrogen donating ability

• DPPH is one of the compounds that possess a c.___________________ with a characteristic absorption, which decreases significantly on exposure to proton radical scavengers

c = ?

inversely proportional

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• the antioxidant property is ________________________ to the coloration of DPPH

512 nm

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• The color changes from purple to yellow after reduction, which can be quantified by its decrease in absorbance at a._____________

• Radical scavenging activity increases with the increasing percentage of b._________________________.

• The c.________________________ indicates the free radical scavenging potential of the sample.

a = ?

free radical inhibition

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• The color changes from purple to yellow after reduction, which can be quantified by its decrease in absorbance at a._____________

• Radical scavenging activity increases with the increasing percentage of b._________________________.

• The c.________________________ indicates the free radical scavenging potential of the sample.

b = ?

degree of discoloration

ANTIOXIDANT ASSAY:

Principle of DPPH (1,1-diphenyl-2-prerylhydrozyl)

• The color changes from purple to yellow after reduction, which can be quantified by its decrease in absorbance at a._____________

• Radical scavenging activity increases with the increasing percentage of b._________________________.

• The c.________________________ indicates the free radical scavenging potential of the sample.

c = ?

standard solution, plant extracts, and blank (methanol)

DPPH ASSAY:

1. In a 96-microwell plate, pipette 100 mcL of ________________________________________________ in triplicates

2. Add 100 mcL of _________________ and incubate for 30 minutes in a dark environment.

3. Read the absorbance at ___________________

1 = ?

DPPH solution

DPPH ASSAY:

1. In a 96-microwell plate, pipette 100 mcL of ________________________________________________ in triplicates

2. Add 100 mcL of _________________ and incubate for 30 minutes in a dark environment.

3. Read the absorbance at ___________________

2 = ?

512 nm

DPPH ASSAY:

1. In a 96-microwell plate, pipette 100 mcL of ________________________________________________ in triplicates

2. Add 100 mcL of _________________ and incubate for 30 minutes in a dark environment.

3. Read the absorbance at ___________________

3 = ?