Antioxidant Assay Principle & Process (DPPH & H2O2): Dr. Bhushan P Pimple

Summary

This e-lecture focuses on in-vitro screening methods used to assess the antioxidant activity of natural products. Dr. Bhushan Pimple explains how antioxidants help prevent oxidative damage caused by free radicals in the body. The lecture covers various methods like the DPPH free radical scavenging assay and hydrogen peroxide scavenging assay to quantify antioxidant potential. The processes involve measuring the decrease in color intensity and absorbance when antioxidants interact with free radicals. Practical examples using natural compounds, such as fruit juices and vitamins, are discussed, along with standard methods for evaluating antioxidant activity in a laboratory setting.

Takeaways

• 😀 Antioxidants play a vital role in protecting the body from damage caused by free radicals produced during oxidation.
• 😀 Free radicals can damage cells, tissues, and organs, and antioxidants stabilize these radicals by donating electrons to prevent further damage.
• 😀 Antioxidants are commonly found in fruits, fruit juices, vitamins, and dietary supplements, and include compounds like vitamin C, vitamin E, and thiols.
• 😀 Antioxidants can also be enzymes produced by the body, such as catalase, glutathione, and superoxide dismutase, which help neutralize oxidative stress.
• 😀 Scavenging free radicals, also known as free radical cleaning, is a key function of antioxidants to protect the body from oxidative damage.
• 😀 The DPPH Free Radical Scavenging Assay is one of the most popular methods used to measure the antioxidant activity of natural products.
• 😀 In the DPPH assay, antioxidants donate electrons to stabilize DPPH free radicals, causing the purple color of the solution to fade, which is directly related to antioxidant activity.
• 😀 In the DPPH assay, the antioxidant activity is inversely proportional to the absorbance: the more antioxidants present, the less the solution absorbs light.
• 😀 The Hydrogen Peroxide Scavenging Assay involves measuring the decrease in absorbance of hydrogen peroxide solutions, which is also influenced by the presence of antioxidants.
• 😀 Both DPPH and Hydrogen Peroxide Scavenging Assays can be quantified using spectrophotometric analysis at specific wavelengths (570 nm for DPPH and 230 nm for hydrogen peroxide).

Q & A

What is the primary topic discussed in the video?

The video discusses in-vitro screening methods and their applications for evaluating natural products, focusing particularly on antioxidant properties.

What is the role of antioxidants in the body?

Antioxidants prevent damage to the body caused by free radicals, which are produced through oxidation. They stabilize free radicals by donating electrons and thus prevent cellular damage.

What is a free radical, and why is it harmful to the body?

A free radical is an atom or molecule with an unpaired electron, which makes it highly reactive. It can steal electrons from healthy molecules, damaging cells, tissues, and organs.

How do antioxidants neutralize free radicals?

Antioxidants neutralize free radicals by donating electrons to them, thereby stabilizing the free radicals and preventing them from causing further damage to the body.

What are some examples of antioxidants commonly used?

Examples of antioxidants include vitamin C, vitamin E, thiols, and enzymes like catalase and glutathione, which are naturally found in the body.

What is the DPPH free radical scavenging assay used for?

The DPPH free radical scavenging assay is used to measure the antioxidant potential of a substance by observing its ability to neutralize DPPH free radicals. The intensity of the purple color in the solution decreases as the antioxidants donate electrons to DPPH.

How does the DPPH assay measure antioxidant activity?

In the DPPH assay, a purple-colored DPPH solution is mixed with an antioxidant. As the antioxidant donates hydrogen to DPPH, the purple color fades, and the decrease in absorbance is used to measure antioxidant activity.

What is the principle behind the hydrogen peroxide scavenging assay?

The hydrogen peroxide scavenging assay measures the ability of antioxidants to reduce the intensity of hydrogen peroxide's color. The antioxidant compounds interact with hydrogen peroxide, leading to a decrease in its absorbance, which indicates their scavenging activity.

How is the hydrogen peroxide scavenging assay conducted?

In this assay, hydrogen peroxide is mixed with a sample, a standard, or a blank, along with a phosphate buffer. After incubation, the absorbance is measured at 230 nm, and the decrease in absorbance indicates the antioxidant activity of the sample.

What is the significance of the control and blank test tubes in these assays?

The control test tube contains hydrogen peroxide and phosphate buffer but no antioxidant, providing a baseline for measuring the absorbance. The blank test tube contains only the buffer solution to account for any absorbance of the buffer itself.