Phytochemistry II: Antioxidants and Free Radicals

10 Questions

What is the wavelength at which the absorbance is measured in the DPPH assay?

517 nm

What is the result of the radical being neutralized in the DPPH assay?

It is converted into a reduced form

What is the purpose of spraying or dipping the TLC plate with DPPH solution in TLC-DPPH bioautography?

To detect the biological activity of the sample

Which of the following antioxidants is commonly used as an anti-aging agent?

Vitamin C

What is the clinical use of Trihydroxyflavones such as apigenin?

Anticancer

What is the antioxidant that is extracted from the seeds of milk thistle?

Silymarin

Which of the following antioxidants is commonly used as an immunity booster?

Vitamin C

What is the result of the radical accepting an electron from an antioxidant in the DPPH assay?

It is converted into a reduced form

What is the advantage of TLC-DPPH bioautography over other antioxidant assays?

It is a simple, economical, and quick method

Which of the following antioxidants is commonly used as a cardioprotective agent?

Lipoic acid

Study Notes

Antioxidants

Antioxidants are substances that prevent or slow damage to cells caused by free radicals, unstable molecules produced by the body as a reaction to environmental and other pressures.
Free radicals are waste substances produced by cells as the body processes food and reacts to the environment, and can cause oxidative stress, leading to cell and body function harm.

Classification of Antioxidants

Natural Antioxidants: Enzymatic (e.g., glutathione peroxidase, superoxide dismutase, and catalase) and Non-Enzymatic (e.g., uric acid, lipoic acid, bilirubin, glutathione, and melatonin)
Exogenous Antioxidants: Synthetic (e.g., butylated hydroxytoluene (BHT), octyl gallate (OG), butylated hydroxyanisole (BHA), propyl gallate (PG), and tert-butylhydroquinone (TBHQ))

Mechanism of Action

Antioxidants directly scavenge ROS or indirectly act by inhibiting ROS production.
Mechanisms include chelating metal catalysts, activating antioxidant enzymes, inhibiting oxidases, and preventing radical chain reactions of oxidation.
Hydrogen atom transfer (HAT) or single electron transfer (SET) mechanisms, followed by proton transfer (SET-PT)

Sources of Natural Antioxidants

Fruits and vegetables (e.g., citrus fruits, kiwi, guava, tomatoes, bell peppers, spinach, broccoli)
Vegetable oils, broccoli, nuts, seeds, egg yolk (Vitamin E)
Fruits (berries, grapes), beverages (tea, cocoa, coffee), vegetables (onions, spinach), spices and herbs (clove bud, turmeric, celery, oregano) (Phenolic antioxidants)
Carrots, sweet potatoes, apricots, pumpkin (Carotenoids)
Meats, shellfish, whole grains (Zinc)
Seafood, meats, whole grains (Selenium)

Antioxidant Assays

1,1-Diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH·)
Ferric ion reducing antioxidant power assay (FRAP)
Oxygen radical absorbance capacity (ORAC)
Total radical trapping antioxidant parameter (TRAP)

DPPH Assay

Measures antioxidant activity by scavenging free radicals
DPPH is a free radical with a deep violet color, which is neutralized by accepting a hydrogen atom or an electron from an antioxidant, converting it to a reduced form (yellow color)
Absorbance is measured at 517 nm using a UV-Visible spectrophotometer

TLC-DPPH Bioautography

A simple, economical, and quick method to screen antioxidant activity
Involves spraying or dipping a developed TLC plate with DPPH solution to give yellow antioxidant spots against a purple background

Clinical Uses of Antioxidants

Anti-aging: Vitamin C, Vitamin E, polyphenolics, and lycopene
Anticancer: Trihydroxyflavones (e.g., apigenin), Epigallocatechin-3-gallate (EGCG), and green tea
Cardioprotective: Lipoic acid and Vitamin C
Hepatoprotective: Silymarin, a mixture of flavonoids extracted from milk thistle seeds
Immunity boosters: Vitamin C and Zinc