antioxidant activity protocol (DPPH assay)

carbomer
1 Jan 201706:08
EducationalLearning
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TLDRThis video script provides a detailed guide on calculating the antioxidant activity of a plant extract using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. DPPH is a purple-colored free radical that, when acted upon by an antioxidant, is reduced to a yellow-colored compound, DPPH-H. The process involves preparing a 0.4 M DPPH solution in methanol, a plant extract at a concentration of 1 mg/mL in a 70:30 methanol to water ratio, and a solvent mixture of ethanol and water. Ascorbic acid is used as a reference due to its high antioxidant activity, with a final concentration of 25 micromolar. The protocol includes making dilutions, exposing the mixture to DPPH for 30 minutes in the dark, and measuring the optical density (OD) at 517 nm. The lower the OD, the higher the antioxidant activity of the plant extract. The video concludes with a formula to calculate the percentage antioxidant activity of the plant extract, which is derived by comparing the OD of the control and the plant extract.

Takeaways
  • 🌿 The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is used to measure the antioxidant activity of plant extracts.
  • 🟣 DPPH is a purple-colored free radical, and its reduction to the yellow-colored DPPH• indicates antioxidant activity.
  • 🛑 A 0.4 M solution of DPPH in 100% methanol should be prepared and protected from light for accurate results.
  • 🍃 Plant extracts should be prepared at a concentration of 1 mg per ml using a 70:30 ratio of methanol to water.
  • 🧪 A 70:30 ratio of ethanol to water is used as the solvent in the assay.
  • 🍋 Ascorbic acid, known for its high antioxidant activity, is used as a reference compound for comparison.
  • 📊 The assay involves making different dilutions of the plant extract and comparing them to ascorbic acid and a control with just DPPH and solvent.
  • ⏱️ The reaction mixture should be kept in the dark at room temperature for 30 minutes before measuring the optical density (OD) at 517 nm.
  • 📉 A higher antioxidant activity in the plant extract is indicated by a lower OD value, as more DPPH is converted to DPPH•.
  • 🧮 To calculate the percentage of antioxidant activity, subtract the OD of the control from the OD of the plant extract, then divide by the OD of the control and multiply by 100.
  • 🔍 The initial concentration of ascorbic acid should be adjusted to maintain a final concentration of 25 micromolar even after dilution with DPPH.
Q & A

  • What does DPP stand for in the context of the video?

    -DPP stands for 2,2-diphenyl-1-picrylhydrazyl, which is a type of free radical used in the DPP essay to measure antioxidant activity.

  • What color is DPPH in its original form?

    -The original form of DPPH is purple.

  • What color does DPPH turn when reduced by an antioxidant?

    -When reduced by an antioxidant, DPPH turns into DPPH-H, which is yellow in color.

  • What is the concentration of DPPH that should be prepared for the assay?

    -The concentration of DPPH that should be prepared is 0.4 M in 100% methanol.

  • What is the solvent ratio used for the plant extract in the video?

    -The solvent ratio used for the plant extract is 70% methanol to 30% water.

  • What is the role of ascorbic acid in the DPPH assay?

    -Ascorbic acid is used as a reference standard in the DPPH assay due to its high antioxidant activity, which allows for comparison with the plant extract's antioxidant activity.

  • What is the final concentration of ascorbic acid used in the assay?

    -The final concentration of ascorbic acid used in the assay is 25 micromolar.

  • How long should the DPPH assay mixture be kept in the dark at room temperature?

    -The DPPH assay mixture should be kept in the dark at room temperature for 30 minutes.

  • At which wavelength is the optical density (OD) measured in the DPPH assay?

    -The optical density (OD) is measured at 517 nanometers in the DPPH assay.

  • How does the optical density (OD) relate to the antioxidant activity of the plant extract?

    -The lower the OD, the more the purple color of DPPH has been converted into the yellow color of DPPH-H, indicating higher antioxidant activity of the plant extract.

  • What is the formula to calculate the percentage antioxidant activity of the plant extract?

    -The percentage antioxidant activity is calculated by taking the OD of the control minus the OD of the respective plant concentration, dividing by the OD of the control, and then multiplying by 100.

  • How do you determine the initial concentration of ascorbic acid needed to achieve a final concentration of 25 micromolar after dilution?

    -You calculate 33% of the desired end concentration (25 micromolar), which is 8.25 micromolar, and add this to the original concentration to compensate for the dilution effect when the DPPH is added.

Outlines
00:00
🌿 Understanding Antioxidant Activity with DPPH Assay

This paragraph introduces the topic of calculating antioxidant activity in plant extracts using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. DPPH is a purple-colored free radical, and its reduction to the yellow-colored DPPH-H is an indication of antioxidant activity. The protocol involves preparing a 0.4 M DPPH solution in methanol, protecting it from light, and using a plant extract prepared at a concentration of 1 mg/mL in a 70:30 methanol-to-water ratio. A solvent mixture of 70:30 ethanol-to-water is also required. Ascorbic acid, known for its high antioxidant activity, is used as a reference compound at a final concentration of 25 micromolar. The process involves making dilutions, exposing the mixture to DPPH for 30 minutes in the dark, and then measuring the optical density (OD) at 517 nanometers. A lower OD indicates higher antioxidant activity, as more DPPH is converted to DPPH-H. The paragraph also explains the calculation of the final concentration of ascorbic acid needed to ensure a 25 micromolar concentration after dilution with DPPH.

05:04
📊 Calculating Percentage Antioxidant Activity

The second paragraph explains how to calculate the percentage antioxidant activity of a plant extract. The process involves subtracting the absorbance of the control (solvent plus DPPH) from the absorbance of the plant extract at a specific concentration, and then dividing this difference by the absorbance of the control. This quotient is then multiplied by 100 to get the percentage antioxidant activity. The paragraph emphasizes the importance of using different blanks for different concentrations to account for the optical density given by the plant extract itself. The video concludes with an invitation for viewers to like, subscribe, and ask questions in the comments section if they have any.

Mindmap
Keywords
💡Antioxidant Activity
Antioxidant activity refers to the capacity of a substance to neutralize free radicals, which are molecules that can damage cells and contribute to aging and various diseases. In the video, the focus is on measuring the antioxidant activity of a plant extract, which is crucial for understanding its potential health benefits.
💡DPPH Assay
DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is a widely used method for determining the antioxidant capacity of a substance. The assay involves the reaction of the purple-colored DPPH radical with an antioxidant, which reduces the DPPH to a yellow-colored diphenylpicrylhydrazine (DPH). The video explains how to perform this assay to evaluate the antioxidant properties of a plant extract.
💡Free Radical
A free radical is an atom or molecule with an unpaired electron that seeks to become more stable by reacting with other molecules, potentially causing damage in the process. In the context of the video, DPPH is a type of free radical that is used to test the antioxidant activity of plant extracts.
💡Plant Extract
A plant extract is a substance obtained from plants through various extraction methods, such as solvent extraction. In the video, the plant extract is used as the sample to be tested for its antioxidant activity using the DPPH assay.
💡Methanol
Methanol is an organic compound used as a solvent in the DPPH assay. It is mentioned in the video as part of the preparation of the DPPH solution and the plant extract. Methanol is important for dissolving the substances and facilitating the chemical reactions necessary for the assay.
💡Ascorbic Acid
Ascorbic acid, also known as vitamin C, is a powerful antioxidant with high antioxidant activity. In the video, it is used as a reference substance to compare the antioxidant activity of the plant extract. Ascorbic acid's known antioxidant properties make it a useful benchmark for evaluating the effectiveness of the plant extract.
💡Ethanol
Ethanol is another organic compound used as a solvent in the DPPH assay. The video specifies a 70 to 30 ratio of ethanol to water as the solvent used in the assay. Ethanol plays a role in the dilution process and helps to maintain the proper conditions for the chemical reactions to occur.
💡Concentration
Concentration in the context of the video refers to the amount of a substance, such as the plant extract or ascorbic acid, present in a given volume of solution. The video discusses preparing the plant extract at a concentration of 1 mg per ml and adjusting the concentration of ascorbic acid to ensure a final concentration of 25 micromolar.
💡Optical Density (OD)
Optical density (OD) is a measure of how much light is absorbed or scattered by a substance and is used in the DPPH assay to measure the antioxidant activity. The video describes taking the OD at 517 nanometers, which corresponds to the color change from purple DPPH to yellow DPPH due to the action of the antioxidant.
💡Dilution
Dilution refers to the process of reducing the concentration of a solute in a solution by adding more solvent. In the video, different dilutions are made for the reference and control samples in the DPPH assay to test the antioxidant activity at various concentrations of the plant extract.
💡Percentage Antioxidant Activity
Percentage antioxidant activity is a measure of the effectiveness of an antioxidant relative to a standard. The video outlines a formula to calculate this percentage using the optical density values obtained from the DPPH assay, which helps to quantify the antioxidant capacity of the plant extract.
Highlights

The transcript discusses calculating antioxidant activity of plant extracts using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.

DPPH is a free radical with a purple color that turns yellow (DPH) when reduced by an antioxidant.

A 0.4 M DPPH solution in 100% methanol is prepared and must be protected from light.

Plant extracts are prepared at a concentration of 1 mg/mL in a 70:30 methanol to water ratio.

A 70:30 ethanol to water ratio is used as a solvent in the assay.

Ascorbic acid, known for its high antioxidant activity, is used as a reference in the assay.

Ascorbic acid is used at a final concentration of 25 micromolar for comparison.

Different dilutions are made for the reference and control groups using DPPH.

The control group uses the solvent plus DPPH to measure the absorbance (OD) of undegraded DPPH.

The assay requires a 30-minute incubation period in the dark at room temperature.

The antioxidant activity is determined by measuring the absorbance at 517 nanometers.

Higher antioxidant activity in plant extracts results in lower OD due to more purple DPPH being converted to yellow DPH.

The highest concentration of plant extract with antioxidant activity will have the lowest OD.

The calculation for the percentage of antioxidant activity involves comparing the OD of the control and the plant extract.

The final concentration of ascorbic acid is adjusted based on dilution to maintain a 25 micromolar concentration.

The calculation for the initial concentration of ascorbic acid is detailed to ensure the final concentration remains at 25 micromolar.

The video concludes with instructions on how to calculate the percentage antioxidant activity of the plant extract.

The presenter encourages viewers to like, subscribe, and ask questions in the comments for further clarification.

Transcripts

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Antioxidant ActivityPlant ExtractsDPP EssayAscorbic AcidMethanolEthanolWaterColor ChangeScientific ProtocolBiological ResearchChemical Assay