The Protocol And Role Of Antioxidants In Biological Research

Free radicals,Biological research, DPPH, Health. Biological research,Cancer,Biotechnology, Cell protection, oxidative

What you'll learn

Definition and function of antioxidants: Understand what antioxidants are and how they protect cells from free radicals.

Historical background: Learn about key discoveries and contributions of pioneering scientists in the field of antioxidants.

Mechanisim of free radicals and antioxidants: Comprehend how free radicals are generated and neutralized by antioxidant

Health benefits: Discover how antioxidants reduce disease risk, support immune function, promote healthy skin, and slow aging.

Role in biological research: Understand how antioxidants are used to study oxidative stress, disease mechanisms, and aging.

Practical skills: Perform the DPPH assay to measure antioxidant activity in various samples

Applications in health and research: Apply knowledge to select and evaluate medicinal plants and biological samples for their antioxidant properties.

This course equips you with essential knowledge and practical skills to explore the critical role of antioxidants in health and scientific research.

Requirements

Individuals seeking to enhance their knowledge of healthy living and preventative measures.

Anyone interested in understanding the science behind antioxidants and their impact on the body

Students and professionals in the fields of nutrition, biology and healthcare who want to deepen their understanding of antioxidants.

Researchers curious about the role of antioxidants in biological experimentation.

Enroll today and embark on a journey to unlock the power of antioxidants for a healthier life and a deeper appreciation for the science behind them!

Description

Antioxidants – Their Importance and Biological ResearchChaptersSection 1: Antioxidants – An Overview1.1. What are antioxidants, and how do they combat free radicals?1.2. Historical discoveries of antioxidants and their health benefitsSection 2: Working of Antioxidantso 2.2. Understanding free radicals and their damaging effects2.3. How antioxidants neutralize free radicals and protect cellsSection 3: Importance of Antioxidantso 3.1. Cellular protection and reduced disease risko 3.2. Supporting immune function and anti-aging effectso 3.3. Protection from environmental toxinso 3.4. Promote healthy skin and brain healthSection 4: Crucial Step in Biological Researcho 4.1. Investigating oxidative stress and cellular protectiono 4.2. Exploring disease mechanisms and develop therapieso 4.3. Identifying antioxidant potential in natural compoundso 4.4. Indispensable toolSection 5: Determination of Antioxidantso 5.1. Why DPPH assay is a popular choice for researcherso 5.2. Principle and protocol for DPPH assay to measure antioxidant activitySection 6: What We Learnto 6.1. Recap of antioxidants' vital role in health and disease preventiono 6.2. Importance of antioxidants in food sources and biological researchSection 1: Antioxidants – An OverviewLecture 1.1: What are antioxidants, and how do they combat free radicals?What are antioxidants?Antioxidants are molecules that prevent or slow the damage to cells caused by free radicals, unstable molecules produced by oxidation. Free radicals are unstable molecules that can harm cells and contribute to aging and various diseases. Fruits, vegetables, nuts, seeds and certain beverages like tea are rich sources of antioxidants.They are found in many foods, particularly fruits and vegetables also in supplements. Common examples of antioxidants include vitamins C and E, beta-carotene, Selenium and various phytochemicals like Terpenoids, Flavonoids and Polyphenols etc.There are two main types of antioxidants:1. Endogenous Antioxidants: Produced naturally within the body.Endogenous nonprotein antioxidants. Glutathione, Alpha-lipoic acid (LA), Coenzyme Q, Ferritin, Uric acid and Bilirubin.Endogenous protein antioxidants. Enzymes like Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GPx).Catalase-Fe (III) + H2O2→ compound ICompound I + H2O2→ Catalase-Fe (III) + 2H2O + O22. Exogenous Antioxidants: These are obtained from the diet. Examples include vitamins C and E, beta-carotene and selenium.Example: The working of GlutathioneLiver cleans our body by removing toxic compounds and chemicals.The illustration explains the Glutathione in liver, bonds to the toxins, chemicals and escorts them out of the body.Lecture 1.2: Historical discoveries of antioxidants and their health benefitsAntioxidants and their effects on health has a rich historical background. Now, we are discussing the historical background and the health benefits of antioxidants.Antioxidants and their health benefits has a rich historical background. Human beings have been utilizing fruits, vegetables, nuts to have a healthy, stable life i.e. free from ailments.But now, we are finding what is inside these fruits, vegetables that make us healthy, free from diseases and protect us from cancer as well. To answer this, we will discuss their historical background.Discovery of Antioxidants:Antioxidants began to emerge in the early 20th century when researchers observed that certain substances prevented oxidation of other molecules. One example of such discoveries, was made by British chemist Joseph Lister in the late 19th century when he noticed the antioxidant properties of thiol compounds.Vitamin C:The Hungarian biochemist Albert Szent-Györgyi conducted revolutionary research on vitamin C (ascorbic acid) and its role as an antioxidant. He also discovered that vitamin C could prevent scurvy, a disease that was caused by vitamin C deficiency.Example: Vitamin C is also known as Ascorbic acid. Here in this illustration you will learn about the sources of Vitamin C, its deficiency results into what diseases and what are the beneficial properties of Vitamin C.The rich sources of Vitamin C include oranges, lemon, kiwi, bell pepper, citrus fruits, strawberries, Brussel sprouts, broccoli and tomato etc.Research has examined the effects of Vitamin C on various health conditions and processes, in the body like cardiovascular diseases, cancer, immune function, skin health and joint pain etc. They came to know this vitamin helps liver and heart functioning, improves our immune system and promote wound healing. Whereas, deficiency of Vitamin C results into dry splitting hair, joint pain, unhealthy skin decline in cognitive properties, slow wound healing, bleeding gums and tooth loss.Vitamin E:The discovery of this antioxidant dates back to the 1920s when researchers Herbert Evans and Katherine Bishop identified a dietary factor necessary for reproduction in rats. This component was later named "tocopherol," derived from the Greek words meaning "to bear offspring,". Later, it was found to have potent antioxidant properties.Example:Vitamin E is a very important antioxidant. Its sources include bell pepper, green olives, papaya, cooked spinach, almonds, pine nuts and sunflower seeds etc. Vitamin E is best for eye health. This Vitamin neutralize free radicals, reduce cancer risk, Alzheimer’s disease, Parkinson disease and helps in fetal development. It also helps in skin treatment, hair growth, cholesterol balance, hormones balance and improves muscular strength.Free Radical Theory of Aging:· From reactive oxygen species to death – Free radicals are also known as reactive oxygen species (ROS). These species can cause damage to lipids, cell membrane, DNA and ultimately death of cells. But can this damaged caused by the reactive oxygen species lead to gaining. Reactive oxygen species (ROS) affect the process of aging due to increased oxidative damage to the biological molecules and the promotion of cellular senescence. Aging involves a systematic loss of functioning with respect to time, in a time dependent manner.· Free radical theory of aging- Research was performed to study the role of free radical in promotion of aging process. This theory was proposed by Denham Harman in the 1950s. He suggested free radicals caused cumulative damage to the normal body cells and resulted aging and age-related diseases. Whereas antioxidants were proposed as a potential barrier to mitigate this damage.Actually what happens, in aging process, free radicals cause oxidative damage to the cells.This damage promotes cellular death. Antioxidants turn on genes to slow down aging process and also by stopping free radicals they stop this chain reaction.Antioxidants from food:Researchers identified numerous naturally occurring antioxidants in foods till now. Fruits, vegetables, nuts, seeds and certain beverages like tea are rich sources of antioxidants.They are found in many foods, particularly fruits and vegetables also in supplements. Common examples of antioxidants include vitamins C and E, beta-carotene, Selenium and various phytochemicals like Terpenoids, Flavonoids and Polyphenols etc.Research on antioxidants:Studies have shown that diets high in antioxidant-rich foods are associated with lower risks of chronic diseases such as heart disease, cancer and neurodegenerative disorders. They also found that a diet rich in antioxidant-containing foods is associated with numerous health benefits, including reduced risk of chronic diseases and improved overall health and longevity.Section 2: Working of antioxidantsAntioxidants are helping compounds that reduce or prevent damage caused by free radicals.Formation of free radicals – antioxidant prevent and reduce the damage caused by free radicals. Free radicals are produced through various processes like inflammation, metabolism, smoking, UV exposure, air pollution, ionizing radiations etc.They can damage cells, proteins, DNA leading to aging, cancer, heart disease and neurodegenerative disorders like dementia, Alzheimer’s disease etc.Inflammation – immune cells called macrophages produce free radicals while fighting off invading germs. These free radicals can damage to healthy cells leading to inflammation.Metabolism – free radicals are also produced during metabolic processes that take palce in mitochondria. OH hydroxide is a powerful free radical and results in DNA damage.Smoking – it also results into the production of free radicals i.e. hydroxide ion.Ionizing radiations – gamma radiations, X-rays. These rays cause radiolysis of cellular water producing free radicals i.e. hydroxide ion, interacting with atoms and destroy DNA.Air pollution – the smoke of factories produces O3 and UV- radiations in the air, resulting in the production of hydroxide ion.UV light from sun – UV light results into the production of free radical i.e. hydroxide ion. This free radical can damage to the DNA by interacting with its structure and results into its destruction. This condition results into the death of cells and also mutation that can cause cancer.Free radical effecting healthy cell - This illustration is explaining the harmful effects of free radicals to a normal healthy cell. When free radicals are produced inside a cell, it creates an oxidative stress inside it. These species interact with the cellular components and cause damage to the cell and destroys its structure. Free radicals also cause damage to DNA and leads to cancer. It also destroys protein and results in its denaturation. It also leads lipid peroxidation.Lecture 2.1: Understanding free radicals and their damaging effectsThe “Free radicals” are molecular compounds with one-electron deficiency also denominated impaired electron in their outer orbital, examples of ROS are superoxide anion, hydroxyl radical, and hydrogen peroxide; nitric oxide and peroxynitrite are included in RNS.Free radicals can damage cells, DNA, protein, lipids and also contribute to aging and various diseases such as cancer, cardiac disease and neurogenerative disease like Dementia and Alzehimer’ s disease etc.Oxidative stress is a condition where imbalance between free radicals and antioxidants in the body takes place. This can cause damage to organ, tissues and results in various diseases. You can help your body by living a healthy life style.1- Heart: Free radicals oxidative stress can lead to congenital heart diseases.2- Skin problems: Free radicals results in skin aging, sunburn and Psoriasis.3- Kidney functioning: Oxidative stress result into chronic kidney diseases, Renal grafts and Nephritis.4- Lungs: Oxidative stress effects lungs and results into COPD (Chronic Obstructive Pulmonary) disease, ARDS (Acute Respiratory Distress) syndrome and cancer etc.5- Brain: Brain is also effected and results in Neurogenerative disorders like OCD - Obsessive compulsive disorder, ADHD - Attention Deficit Hyperactivity disorder and stroke, trauma etc.6- Immune System: Oxidative stress effects immune system and results into specific immunoresponse disorder like MS and IBO etc.7- Blood vessels: Oxidative stress effects blood vessels and results into Restenosis, Atherosclerosis, Endothelial Dysfunction and Hypertension etc.8- Oxidative stress effects overall body organs and results into Diabetes, aging and chronic fatigue.9- Oxidative stress effects eye functioning and results into Macular degeneration, Retinal degeneration and Cataract etc.10- Oxidative stress effects joints and results into Rhematoid, Osteo Arthritis and Psorasis etc.Lecture 2.2: How antioxidants neutralize free radicals and protect cellsThese friendly compounds work by neutralizing free radicals hence prevent and also reduce oxidative stress as well as damage to the normal cells.To achieve this, antioxidants simply donate electrons to free radicals, making then stabilize them. After stabilizing they become less harmful.Section 3: Importance of antioxidantsThis section is dealing with the importance of antioxidants. Antioxidant play a vital role in protecting normal cells from oxidative stress. This stress lead to the damage of normal cells, changing its shape, disturbing its homeostasis, damaging DNA, protein and lipids.Lecture 3.1: Cellular protection and reduced disease riskCellular protection:Antioxidants protect cells from oxidative damage. They also prevent cell membranes, proteins, DNA, maintain cellular integrity and improves their functionality.Decrease Disease Risk:Oxidative stress results in the development of many chronic diseases like cancer, cardiovascular disease, diabetes and neurodegenerative disorders. They reduce the risk of these conditions by mitigating oxidative damage and inflammation within the body.Lecture 3.2: Supporting immune function and anti-aging effectsSupport Immune FunctionAntioxidants also protect immune cells from oxidative damage hence support a healthy immune system. They help the body defend against infections and illnesses more effectively.Anti-aging effectsOxidative stress is a major contributor to the aging process. Antioxidants help to reduce aging process by protecting cells and tissues from oxidative damage and promote longevity and vitality.Lecture 3.3: Protection from environmental toxins:Antioxidants reduce the harmful effects of environmental toxins, pollutants such as air pollution, cigarette smoke and ultraviolet radiation.Antioxidants also neutralize the toxins produced by free radicals and reduce their damaging impact on the body.Lecture 3.4: Promote healthy skin and Brain HealthAntioxidants promote healthy skin and healthy brain and also improves its functioning. they does this by protecting neurons and brain cells. Also improves brain thinking ability and decrease the risk of trauma and cancer.Promote healthy skinAntioxidants play a vital role in maintaining healthy skin by protecting against oxidative damage from UV radiation and environmental pollutants. Also prevent premature aging, wrinkles & keep the skin looking youthful and radiant.Improves brain healthOxidative stress results in cognitive decline and neurodegenerative diseases such as Alzheimer's and Parkinson's. They protect brain cells from oxidative damage and may also support cognitive function and memory as we age.Section 4: Crucial step in biological researchLecture 4.1: Investigating oxidative stress and cellular protectionOxidative Stress investigation:Oxidative stress is a state where the balance between free radicals and antioxidants is disrupted, leading to cellular damage.Researchers perform antioxidants assay to check oxidative stress levels in experimental models. This also help them to study its effects on cellular function, signaling pathways also the gene expression.Cellular Protection:During experiments, antioxidants are used to protect cells and tissues from oxidative damage.Researchers do this by adding antioxidants to cell cultures or animal models, this step reduces the harmful effects of reactive oxygen species (ROS), maintain cellular integrity, and ensure accurate experimental results.Lecture 4.2: Exploring disease mechanisms and develop therapiesSome diseases are associated with oxidative stress e.g., cancer, cardiovascular diseases and neurodegenerative disorders.Antioxidants assay is used to investigate the role of oxidative damage in disease progression and to explore potential therapeutic strategies targeting antioxidant pathways.Study Aging Processes:Researchers use this assay to understand how oxidative damage accumulates over time and effects on cellular senescence, DNA damage, mitochondrial function and provide insights into the biology of aging.Develop Therapies:Antioxidants has therapeutic potential for various conditions that are resulted from oxidative stress.Biological researchers perform antioxidant assay to explain the mechanisms of action of antioxidants and also optimize their efficacy for therapeutic use.Also they investigate the effect of antioxidants as a standalone treatments or need other therapies in combination with existing interventions.Lecture 4.3: Identifying antioxidant potential in natural compoundsBiological researches often perform antioxidant assay to evaluate the antioxidant potential of compounds. e.g., plant extracts, synthetic molecules and pharmaceutical agents.Antioxidant assays determine the ability of these natural compounds to neutralize free radicals and also prevent oxidative damage in biological systems.This easy and small step also helps in the identification of potential therapeutic agents against chronic diseases, hence saves time and resources as well.Lecture 4.4: Indispensable tool• Unraveling the complexities of oxidative stress• Observing and investigating disease mechanisms• Developing novel therapeutic strategies to promote health• Combat oxidative damage-associated conditionsSection 5: Determination of antioxidantsLecture 5.1: Why DPPH assay is a popular choice for researchersDPPH is discovered by Goldschmidt and Renn in 1920s. It is a stable free radical with purple color which turns yellow when scavenged. The is relatively rapid, fast, quick and cost-effective that measure overall antioxidant activity.The DPPH assay is relatively rapid, fast, quick and cost-effective that measure overall antioxidant activity.Even weak antioxidants give results with 30 min incubation with this radical.Multiple samples can be screened simultaneously and get the results.The visual change of DPPH color from purple to yellow gives a rough estimation of antioxidant potential of our samples even without spectrophotometer.This test must be used as preliminary, crucial and helpful in plant selection. If your plant has significant antioxidant value, it can be the best choice to cure diseases caused by free radicals like cancer, Alzheimer’ s etc.Hence it a popular choice for researchers for screening of the antioxidant activity of various compounds, natural extracts and food samples and get rapid results.Lecture 5.2: Principle and protocol for DPPH assay to measure antioxidant activityPrincipleAntioxidants reacts with DPPH that changes its color from purple to yellow upon reduction.ProtocolA- Preparation of SolutionsSample solution0.5 mg/mL sample solution is prepared in ethanol (A).DPPH solution0.1mM DPPH solution is prepared in ethanol (B). This is deep purple colored solution, save it in dark colored bottle.Standard solution0.5 mg/mL Trolox (Vit E analogue) solution prepared in ethanol.Control Solution3 mL of DPPH solution.Blank SolutionEthanol is used as a blank.B: Rapid PerformanceTake 1 mL of sample solutions and standard solution separately in already labelled sterilized glass test tubes.Add 2 mL of DPPH solution (B) in sample solution (A) and in standard solution simultaneously.Add 3 mL of DPPH solution in a test tube and label it as control.Now leave them for 30 min incubation (in dark).After incubation, adjust UV-vis spectrophotometer absorbance at 517 nm place and place blank to set baseline.Start taking absorbance of the samples, standard and control one by one.The percentage inhibition or scavenging activity of the samples will be calculated using the following formula:%Inhibition of DPPH= [(Bcontrol–Asample)/Bcontrol] × 100Where:B Control ​ is the absorbance of the DPPH solution (DPPH + Ethanol).A Sample ​ is the absorbance of the DPPH solution + sample solution.Results of the samples will be compared with standard. Any increase or decrease in percentage DPPH inhibition will tell us the significance of our samples.Section 6: What We LearntLecture 6.1: Recap of antioxidants' vital role in health and disease prevention· Antioxidants play a vital role in maintaining overall health and well-being.· They protect cells from oxidative damage caused by free radicals.· They are essential for reducing the risk of chronic diseases, supporting immune function, promoting skin health and potentially slow down the aging process.Lecture 6.2: Importance of antioxidants in food sources and biological research· They are found naturally in a variety of foods, fruits, vegetables, nuts, seeds and whole grains.· Antioxidant determination helps in medicinal plant selection for any biological activity.

Overview

Section 1: Course Contents

Lecture 1 Course contents

Lecture 2 Course Contents Cont.

Section 2: Antioxidants – An Overview

Lecture 3 Antioxidants – An Overview

Lecture 4 1.1. What are antioxidants, and how do they combat free radicals? Part 1

Lecture 5 1.1: What are antioxidants, and how do they combat free radicals? Part 2

Lecture 6 1.1: What are antioxidants, and how do they combat free radicals? Part 3

Lecture 7 1.1. What are antioxidants, and how do they combat free radicals? Part 4

Lecture 8 1.2: Historical discoveries of antioxidants and their health benefits Part 1

Lecture 9 1.2: Historical discoveries of antioxidants and their health benefits Part 2

Lecture 10 1.2: Historical discoveries of antioxidants and their health benefits Part 3

Lecture 11 1.2: Historical discoveries of antioxidants and their health benefits Part 4

Lecture 12 1.2: Historical discoveries of antioxidants and their health benefits Part 5

Lecture 13 1.2: Historical discoveries of antioxidants and their health benefits Part 6

Lecture 14 1.2: Historical discoveries of antioxidants and their health benefits Part 7

Section 3: Section 2: Working of antioxidants

Lecture 15 2.0 Working of antioxidants Part 1

Lecture 16 2.0 Working of antioxidants Part 2

Lecture 17 2.1: Understanding free radicals and their damaging effects Part 1

Lecture 18 2.1: Understanding free radicals and their damaging effects Part 2

Lecture 19 2.2: How antioxidants neutralize free radicals and protect cells

Section 4: Section 3: Importance of antioxidants

Lecture 20 3.0: Importance of antioxidants

Lecture 21 3.1: Cellular protection and reduced disease risk Part 1

Lecture 22 3.1: Cellular protection and reduced disease risk Part 2

Lecture 23 3.2: Supporting immune function and anti-aging effects

Lecture 24 3.3: Protection from environmental toxins

Lecture 25 3.4: Promote healthy skin and Brain Health Part 1

Lecture 26 3.4: Promote healthy skin and Brain Health Part 2

Section 5: Section 4: Crucial step in biological research

Lecture 27 4.1: Investigating oxidative stress and cellular protection Part 1

Lecture 28 4.1: Investigating oxidative stress and cellular protection Part 2

Lecture 29 4.2: Exploring disease mechanisms and develop therapies Part 1

Lecture 30 4.2: Exploring disease mechanisms and develop therapies Part 2

Lecture 31 4.3: Identifying antioxidant potential in natural compounds

Lecture 32 4.4: Indispensable tool

Section 6: Section 5: Determination of antioxidants

Lecture 33 5.1: Why DPPH assay is a popular choice for researchers

Lecture 34 5.2- Principle & protocol for DPPH assay to measure antioxidant activity Part 1

Lecture 35 5.2- Principle & protocol for DPPH assay to measure antioxidant activity Part 2

Section 7: Section 6: What We Learnt

Lecture 36 6.1: Recap of antioxidants' vital role in health and disease prevention

Lecture 37 6.2: Importance of antioxidants in food sources and biological research

Lecture 38 Feedback

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