Dear Sandeep,

Hydroxyl radical scavenging activity

Hydroxyl radical is one of the potent reactive oxygen species in the biological system. It reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids and causes damage to cell (Halliwell and Gutteridge, 1981).

Principle

HRS assay is used to find the scavenging activity of free hydroxyl radicals (which

damage the body cells) like hydrogen peroxide in the presence of different concentrations of plant sample. The model used is ascorbic acid-iron-EDTA model of hydroxyl radical generating system. This is a totally aqueous system in which ascorbic acid, iron and EDTA conspire with each other to generate hydroxyl radicals.

Reagent preparation

 Iron-EDTA solution was prepared by mixing 0.13% ferrous ammonium sulphate and 0.26% EDTA.

 0.018% EDTA was prepared by dissolving 0.018g EDTA in 100ml dist. H2O.

 0.22% ascorbic acid was prepared by dissolving 0.22g ascorbic acid in 100ml dist. H2O.

 17.5% TCA was prepared by dissolving 17.5g TCA in 100ml of dist.H2O.

 Nash reagent was prepared by adding 7.5g ammonium acetate, 0.5ml of glacial acetic acid and 0.2ml of acetone to 100ml dist.H2O.

Working procedure

Various concentrations of extract (250, 500, 750, 1000µg) were taken and 1ml of iron EDTA solution, 0.5ml of EDTA solution, 1ml of DMSO and 0.5ml of ascorbic acid were added to it. The mixture was incubated in a boiling water bath at 80 to 90°C for 15 min. After incubation, 1ml of ice cold TCA and 3ml of Nash reagent were added and the reaction mixture was incubated at room temperature for 15 min. The absorbance was read at 412 nm.

The % hydroxyl radical scavenging activity is calculated by the following formula,

 SEE ATTACHED FILE

Where, HRSA is the Hydroxyl Radical Scavenging Activity, Abs control is the absorbance of control and Abs sample is the absorbance of the extract.

For more on this topic, please see attached file.

Hoping this will be helpful,

Rafik

Hydroxyl radical reacts with almost all organic molecules with a diffusion controlled rate constants. This means that all organic molecules have the same antioxidant (radical scavenging) activity towards hydroxyl radical. 

Dear Rafik,

You wrote that "HRS assay is used to find the scavenging activity of free hydroxyl radicals (which damage the body cells) like hydrogen peroxide." Did you mean that free hydroxyl radicals damage the body cell like hydrogen peroxide? 

Hi Mr. Karaman  thanks for your guide . i will go ahead with this protocol. 

Dear Yurii,

Free hydroxyl radicals are believed to damage the body cells. On the other hand, hydrogen peroxide is used in the HRS assay to determine the scavenging effecincy of hydroxyl radicals by the chemlcals described in the working procedure depicted in my previous answer.

Regarding the cell damrage by hydrogen peroxide: it was reported that Phenanthroline, a strong iron chelator, prevents both the formation of DNA single-strand breaks and the killing of mouse cells produced by H2O2. These results indicate that the DNA damage is produced by hydroxyl radicals formed when H2O2 reacts with chromatin-bound Fe2+ and that this damage is responsible for the killing effect.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1153333/

Other drawbacks of the use of large quantities of hydrogen peroxide are illustrated in the following publication:

Hydrogen peroxide is an oxidising agent that is used in a number of household products, including general-purpose disinfectants, chlorine-free bleaches, fabric stain removers, contact lens disinfectants and hair dyes, and it is a component of some tooth whitening products. In industry, the principal use of hydrogen peroxide is as a bleaching agent in the manufacture of paper and pulp. Hydrogen peroxide has been employed medicinally for wound irrigation and for the sterilisation of ophthalmic and endoscopic instruments. Hydrogen peroxide causes toxicity via three main mechanisms: corrosive damage, oxygen gas formation and lipid peroxidation. Concentrated hydrogen peroxide is caustic and exposure may result in local tissue damage. Ingestion of concentrated (>35%) hydrogen peroxide can also result in the generation of substantial volumes of oxygen. Where the amount of oxygen evolved exceeds its maximum solubility in blood, venous or arterial gas embolism may occur. The mechanism of CNS damage is thought to be arterial gas embolisation with subsequent brain infarction. Rapid generation of oxygen in closed body cavities can also cause mechanical distension and there is potential for the rupture of the hollow viscus secondary to oxygen liberation. In addition, intravascular foaming following absorption can seriously impede right ventricular output and produce complete loss of cardiac output. Hydrogen peroxide can also exert a direct cytotoxic effect via lipid peroxidation. Ingestion of hydrogen peroxide may cause irritation of the gastrointestinal tract with nausea, vomiting, haematemesis and foaming at the mouth; the foam may obstruct the respiratory tract or result in pulmonary aspiration. Painful gastric distension and belching may be caused by the liberation of large volumes of oxygen in the stomach. Blistering of the mucosae and oropharyngeal burns are common following ingestion of concentrated solutions, and laryngospasm and haemorrhagic gastritis have been reported. Sinus tachycardia, lethargy, confusion, coma, convulsions, stridor, sub-epiglottic narrowing, apnoea, cyanosis and cardiorespiratory arrest may ensue within minutes of ingestion. Oxygen gas embolism may produce multiple cerebral infarctions. Although most inhalational exposures cause little more than coughing and transient dyspnoea, inhalation of highly concentrated solutions of hydrogen peroxide can cause severe irritation and inflammation of mucous membranes, with coughing and dyspnoea. Shock, coma and convulsions may ensue and pulmonary oedema may occur up to 24-72 hours post exposure. Severe toxicity has resulted from the use of hydrogen peroxide solutions to irrigate wounds within closed body cavities or under pressure as oxygen gas embolism has resulted. Inflammation, blistering and severe skin damage may follow dermal contact. Ocular exposure to 3% solutions may cause immediate stinging, irritation, lacrimation and blurred vision, but severe injury is unlikely. Exposure to more concentrated hydrogen peroxide solutions (>10%) may result in ulceration or perforation of the cornea. Gut decontamination is not indicated following ingestion, due to the rapid decomposition of hydrogen peroxide by catalase to oxygen and water. If gastric distension is painful, a gastric tube should be passed to release gas. Early aggressive airway management is critical in patients who have ingested concentrated hydrogen peroxide, as respiratory failure and arrest appear to be the proximate cause of death. Endoscopy should be considered if there is persistent vomiting, haematemesis, significant oral burns, severe abdominal pain, dysphagia or stridor. Corticosteroids in high dosage have been recommended if laryngeal and pulmonary oedema supervene, but their value is unproven. Endotracheal intubation, or rarely, tracheostomy may be required for life-threatening laryngeal oedema. Contaminated skin should be washed with copious amounts of water. Skin lesions should be treated as thermal burns; surgery may be required for deep burns. In the case of eye exposure, the affected eye(s) shod eye(s) should be irrigated immediately and thoroughly with water or 0.9% saline for at least 10-15 minutes. Instillation of a local anaesthetic may reduce discomfort and assist more thorough decontamination.

https://www.ncbi.nlm.nih.gov/pubmed/15298493

Hoping this will clarify my point,

Rafik

Dear Rafik,

My remark to your answer was to certain extent provocative. I wanted to pay your attention that hydroxyl radical and hydrogen peroxide do NOT damage cells in the same way. H2O2 is a pretty inert molecule and requires a catalyst  to produce hydroxyl radical as commonly believed but actually wrong.There are thousand papers on antioxidant and antioxidant assays.  I know very well this area and understand  the chemistry behind each assay. I entered this area about 20 years ago after consulting one of pharma company. As a result I developed my own assay based on hydroxyl radical generation from peroxynitrite self decomposition

Peroxynitrite scavenging by different antioxidants. Part I: convenient assay

GGA Balavoine, YV Geletii

Nitric oxide 3 (1), 40-54

This publication was cited  more than 150 times.

I  like your comments and answers. You always give a detail description how to proceed further. At the same time I disagree with such approach. I prefer to stimulate the students to find the answer themselves by providing some hints where and how to look the answer.  Unfortunately, this forum is flooded by questions which can be easily answered by reading textbooks.  I openly downvote such questions.

Back to the original question. Almost all organic molecules scavenge hydroxyl radical with the same rate constants. An antioxidant able to protect a target molecule from a direct reaction with HO(.) DOES NOT exist.  

Dear Yorii,

Happy thanksgiving to you and my friend Prof. Fred Menger.

Rafik

You may find the protocol we used on hydrogen peroxide scavenging useful perhaps

https://www.researchgate.net/publication/321330966_Antioxidant_Potential_and_Total_Phenolic_Changes_in_Malawian_Herbal_Formulations_Stored_In_Clay_Pots_and_Plastic_Bottles