3.5.2. TBARS Assay

  This protocol uses the TBARS (thiobarbituric acid reactive substances) assay that estimates the concentration of malondialdehyde, a product of lipid peroxidation, in a sample. This assay was used to measure the antioxidant capability of polyphenols in the seed coat extracts to protect the lipid bilayer against peroxidation in liposomes as a simulated model for a biological cell membrane. This assay was conducted according to the method proposed by Subramanian et al [39].

Materials and Reagents

1,1,3,3-tetramethoxypropane (TMP), Sigma Aldrich, US

1-butanol, Sigma Aldrich, US

2-thiobarbituric acid (TBA), Sigma Aldrich, US

Ascorbic acid, Fisher Scientific, Canada

Iron (II) sulfate heptahydrate, Sigma Aldrich, US

Hydrochloric acid, EMD Serono, Canada

Methanol, Fisher Chemical, US

Myricetin-3-O-glucoside, Extrasynthese, France

Sodium dodecyl sulfate (SDS), Sigma Aldrich, US

Soy phosphatidyl choline (PC), Sigma Aldrich, US

Trichloroacetic acid (TCA), Sigma Aldrich, US

Equipment

Thermomixer

Vortex

Glycol bath

Centrifuge

Zetasizer (Malvern)

UV-Vis spectrophotometer (plate reader option)

Software

Microsoft Excel 

Procedure

A. Preparation of empty liposomes:

1. Dissolve 200 mg of soy phosphatidyl choline in 50 mL of ethyl acetate to dissolve the lipid (Note 1).
2. Evaporate ethyl acetate from the lipid solution prepared in the previous step using a rotary evaporator to get a uniform thin dried lipid layer on the rotary flask walls.
3. Re-dissolve the thin lipid layer in 100 mL warm water, then sonicate the formed solution for 1 h at 55°C to form unilamellar vesicles as shown in Figure A.

Figure A: The structure of unilamellar vesicle entrapping aqueous solution inside

     4. Measure the particle size of formed unilamellar liposomes using Malvern zetasizer (it should be within 115 ± 15 nm). The smaller the liposome size, the greater the surface area exposed to peroxidation.

     5. Add 100 mL of warm water to the liposome solution to bring it to 1 mg/mL as final concentration.

B. Antioxidant activity measurement using TBARS assay

1. For samples, add 200 µL of liposome solution (1 mg/mL) to 40 µL of diluted extracts (in 10% methanol) and shake for 15 min (Note 2).
2. For control, add 200 µL of liposome solution (1 mg/mL) to 40 µL of 10% methanol and shake for 15 min.
3. For standards, add 200 µL of liposome solution (1 mg/mL) to 40 µL of standard solutions (in 10% methanol) and shake for 15 min.
4. For blank, add 240 µL of 10% methanol (no liposomes).
5. To initiate lipid oxidation, add 40 µL of each of the following solutions: 0.5 mM FeSO₄ solution, 5 mM ascorbic acid and water into the blank/control/sample /standard solutions.
6. Put these mixture solutions in a Thermomixer for shaking and incubation at 37ºC for 2 h.
7. Add 40 µL of 3% sodium dodecyl sulfate and 400 µL of TCA/TBA/HCl solution, then vortex.
8. Put these solution mixtures in a glycol bath at 95ºC for 30 min to allow TBA to react with any lipid peroxidation products.
9. Take these solutions out of the bath and let them cool down.
10. Add 800 µL of 1-butanol to each reaction mixture, vortex for 20 sec then centrifuge at a speed of 16,200g for 10 min (2 layers appear; upper is organic and lower is aqueous). 
11. Transfer 200 µL of the organic (upper) layer of each reaction mixture to a 96-well plate.
12. Read the absorbance of solution mixtures in 96-well plate using a microplate reader at 532 nm.

Data analysis

1. First, absorbance readings of all control/sample/standard should be subtracted from blank reading.
2. Using Excel, plot a calibration curve of TMP solution concentrations vs their absorbance readings (subtracted from blank) and get the slope and intercept (Note 3).
3. Calculate the % inhibition of lipid peroxidation using the following equation (Note 4):

    4. Since each extract sample has a series of dilutions, the % inhibition is calculated for each dilution. The calculated % inhibition are used to calculate the concentration of each extract (µg/mL) required to inhibit 50% of the formation of lipid peroxidation product in the reaction mixture (IC₅₀) using an online IC₅₀ calculator tool.

    5. Calculate the antiradical power (ARP) by getting the reciprocal of IC₅₀, so results can be easily compared.

Notes

Note 1: Liposomes, simulating cell membranes in a biological system, are considered as a suitable substrate for measuring the oxidation of polyunsaturated lipids. 

Note 2: Serial dilutions of each extract solution should be prepared and used in this experiment. The % inhibition of these dilutions are needed to calculate the IC₅₀ for each extract. There is no exact dilution to be used. It is totally dependent on trials to check which concentration range could help to protect liposomes against lipid peroxidation.

Note 3: This calibration curve should be linear (R² should be higher than 0.9).

Note 4: All absorbance readings used in the % inhibition calculation should be subtracted from blank readings.

Preparations

• Ascorbic acid (5 mM): 88 mg of ascorbic acid powder in 100 mL water. 
• Ferrous sulfate (0.5 mM): 14 mg of ferrous sulfate heptahydrate powder in 100 mL water. 
• Sodium dodecyl sulfate (SDS, 3% w/v): 1.5 g of sodium dodecyl sulfate in 50 mL water.
• 15% TCA/ 0.375% TBA/0.25 M HCl mixture: 30 g of trichloroacetic acid (TCA) + 0.75 g of thiobarbaturic acid (TBA) in 195.8 mL of water. Then add 4.2 mL of 12 M HCl.
• TMP standard solution (stock 100 μg/mL): 0.1 mL of TMP in 10 mL of methanol then dilute this solution into serial dilutions in the range of 2-55 μg/mL. For example, take 100 μL of TMP stock solution and mix it with 900 μL of 10% methanol to give a final concentration of 10 μg/mL.

References

Subramanian, M., G.J. Chintalwar, and S. Chattopadhyay, Antioxidant and radioprotective properties of an Ocimum sanctum polysaccharide. Redox Report, 2005. 10(5): p. 257-264.

Farvin, K.H.S. and C. Jacobsen, Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chemistry, 2013. 138(2-3): p. 1670-1681.

Poudel, A., et al., Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives. Pharmaceutics, 2019. 11(4).

AAT Bioquest, I. Quest Graph™ IC50 Calculator. 2020  May 1, 2020; Available from: https://www.aatbio.com/tools/ic50-calculator.