The standard for Beta carotene/linoleic acid bleaching assay is to measure the antioxidant activity of a substance by monitoring its ability to inhibit the oxidation of beta-carotene and linoleic acid in a model system. This assay is commonly used to evaluate the antioxidant potential of natural products, such as plant extracts or essential oils. The standard procedure involves preparing a reaction mixture containing beta-carotene, linoleic acid, and the test substance, and then monitoring the decrease in absorbance at a specific wavelength over time. The antioxidant activity is determined by comparing the rate of bleaching in the presence and absence of the test substance.

The Beta Carotene/Linoleic Acid Bleaching Assay is a popular technique for determining antioxidant activity of substances by assessing their capacity to block beta-carotene oxidation in the presence of linoleic acid. The following are the assay guidelines and possibilities.

Beta Carotene/Linoleic Acid Bleaching Assay Standards

Positive Control: Common standards used in this test include butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). They are synthetic antioxidants with known antioxidant activity. These standards are used to assess the inhibition of beta-carotene bleaching.

BHT/BHA substitutes

Antioxidants found in nature: Natural substances can be utilized as positive controls instead of synthetic antioxidants. Natural antioxidants with demonstrated efficiency include vitamin C (ascorbic acid) and vitamin E (alpha-tocopherol). Their antioxidant activity can be compared to the samples being tested.

Extracts of plants: Plant extracts high in antioxidants can also be used as positive controls. Green tea, rosemary, and grape seed extracts, for example, which contain polyphenols with antioxidant qualities, can be used as substitutes.

Trolox (Analog of Water-Soluble Vitamin E): Trolox, a water-soluble vitamin E derivative, is another possible positive control. It is commonly used in antioxidant tests and can be used as a control molecule in the Beta Carotene/Linoleic Acid Bleaching Assay.

Thank you Rizwan Mehmood Bhatti for the answer. Do you know what is the blank used for the assay? And the negative control that can be used?

Farah Fatihah

The implementation of a blank and a negative control in the Beta Carotene/Linoleic Acid Bleaching Assay is critical for appropriate interpretation of data. The blank is used as a baseline to control for non-antioxidant-related color changes, such as solvent effects or beta-carotene decay. The blank typically includes all assay components except the test sample or antioxidant. By measuring the absorbance of the blank at the start of the test, any variations in subsequent readings may be ascribed to antioxidant activity specifically.

The negative control, on the other hand, is critical in creating a reference point for measuring the efficiency of test materials in suppressing beta-carotene oxidation. The negative control is intended to have little or no antioxidant activity, serving as a baseline against which the antioxidant capacity of the test samples may be evaluated. A negative control is typically made up of a solution comprising solely the solvent used to dissolve the test items. In the negative control, for example, ethanol or acetone might be used as the solvent. This assures that any suppression of beta-carotene oxidation detected is due to the test sample's antioxidant characteristics rather than the solvent itself.

Selecting an adequate negative control is critical in appropriately measuring an element's antioxidant capability. It assures that any variations in absorbance in the environment of the test sample are due to its antioxidant potential rather than being influenced by the negative control's intrinsic qualities. It also enables for the comparison of the test sample to a standardized reference, which aids in determining the sample's relative antioxidant efficacy.

Thank you again Rizwan Mehmood Bhatti !

Just for a confirmation, the blank is the

tween-linoleic acid-chloroform (after chloroform evaporation) + addition of distilled water, am I right?

After reading the absorbance for blank and different concentrations of positive control, is this the formula that will be applied?

% =(Absorbance after 2 hours/ Initial Absorbance)*100

Farah Fatihah The blank in the Beta Carotene/Linoleic Acid Bleaching Assay is normally the reaction mixture without any test sample or antioxidant. It contributes to the explanation of any color changes or absorbance variations that are unrelated to antioxidant activity. The tween-linoleic acid-chloroform combination is normally poured to the blank after the chloroform has evaporated, and then distilled water is added.

As a result, your understanding is accurate. After evaporation, the tween-linoleic acid-chloroform combination is commonly used as the blank, followed by the addition of distilled water. This serves as a baseline for measuring initial absorbance and aids in the correction of any non-specific changes in color or absorbance during the experiment.

And, yes you're on the correct road with your formula. The percentage inhibition (% inhibition) in the Beta Carotene/Linoleic Acid Bleaching Assay is usually computed using a formula similar to the one you provided:

This formula expresses the antioxidant activity as a percentage decrease in absorbance. A larger proportion suggests better beta-carotene oxidation inhibition, signifying stronger antioxidant capabilities.

Simply confirm that the absorbance after 2 hours and Initial Absorbance values acquired from your experimental results are valid.

Thank you Rizwan Mehmood Bhatti !! I appreciate your help and guidance, they will definitely help me a lot in my Final Year Project. Thank you ^^