Biotech Practical Copy

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BIOTECH LAB MANUAL-10TH SEM 2012

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Experiment No#1To evaluate Anti-bacterial activity of a selected Bacterial specie

Introduction:

The process of placement of culture cell to reproduce it in a specificenvironment to make it viable (that can reproduce).the item is inoculated is

known as inoculum.

Viable:

Those species which have ability to reproduce are called viable while whichdont reproduce are non-viable like dust particle.

Incubation:

A process of growth /reproduction of culture at most favorable temperaturein an incubator.

Strain:

Strain it is the lowest taxonomic rank, strain is actually the subtype of specievaries during replication. Strain is identified on the basis of genetic studies.

Mutation:

Genetic variation is known as mutation which may be physical or chemicalchange or due to stress.

Stock solution:

It is a solution (concentration) which must be diluted prior to use.Antibiotics:

Organic compounds produced by a group of micro-organisms or a singlemicro-organism and are used to kill other micro-organisms.

Classification of Antibiotics:

On the basis of their Action

Bacteriostatic: Bactericidal:

Agents which inhibit further growth of micro-

organism e.g. sulphonamides .

They kill the microorganisms as the

bacteriostatic inhibits the growth while

bactericidal are effective when microbes are

replicating, so these drugs are not given in

combination

On the basis of spectrum of activity

Narrow spectrum

antibiotics

Broad spectrum antibiotics Extended spectrum

They are effective against

limited number of micro-

organism e.g. benzyl

penicillin, benzathene

penicillin.Amino-glycosides

are active only against Gram

positive species.

They are effective against wider

range of microorganism,

cephalosporin amoxicillin,

erythromycin, clarithromycin are

active against both gram positive

and gram negative species.

These are blood spectrum

antibiotics even effective

against Gram negative

resistant species. E.g.

Pseudomonas aeruginosa.

It is not treated by some

broad spectrum agents

due to its resistance and

genetic make up


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Classification of Bacteria:

PsychrophilesThey grow below 10 C or 15 c .they are found on glaciers.

Mesophiles

They survive between 25c to 45c and found in plains of Pakistan.

ThermophilesThey survive at temperature higher than 40 c but less than 80c i.e. in deserts

and higher areas of Pakistan.

Extreme Thermophiles They can grow above 80c and they can even grow on hot

boiling water e.g. geysers.


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Experiment No # 2To evaluate the antibacterial activity of selective bacterial species

screening methodMethods

Cross streak method Stabbing method Inoculation method

Streaking is a method of purification of colonies.

Requirements Producer strains Indicator strains

Producer strains

Following are the strains which are evaluated for their capacity of producingantibiotics.

Bacillus subtilis (gram +ve) E.coli (gram-ve)

They are reported to produce bacitracin and subtilin (peptide antibiotic) which

inhibit cell wall synthesis. E.coli is not able to produce antibacterial activity.

Indicator strainFollowing are the strains against which antibiotic producing strains will be tested

Staphylococcus aureus Micrococcus luteus E .coli

Other Requirements

Wire loop Inoculation loop Cotton swab Nutrient agar plate Chloroform Filter paper Borer 6-8 mm in diameter.

Procedure1. Prepare nutrient agar plate and incubate at for 24 hours at 37c.(sterility

check)

2. Refresh cultures of both the indicator and the producer strain.3. Take the nutrient agar plate and draw a straight line of indicator strain in

the center of plate with the help of wire loop.

4. With a gap of 1-2 mm from central line draw a line of the indicator strain.5. Incubate the plate at 37c for 24 hours.6. Then check the results.


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ResultPositive results:

A zone of inhibition will be observed near the junction of producer an

indicator strain

Negative Results:

No zones of inhibition are observed

AdvantageThe method is useful to evaluate more than one test strain against single indicator

strain.

We can also perform this test reversely with one test strain and more than one

indicator strain.

A. Stabbing method1. Prepare nutrient agar plate.2. Incubate it for 24 hours at 37c to check sterility.3. Refresh cultures of both procedure and indicator strain by streaking

method.

4. Incubate culture for 24 hours at 37c.5. Take nutrient broth in test tube.6. Now take test strain by the help of inoculating needle and place it within

depth of nutrient agar plate. Incubate nutrient agar for 24 hours at 37c.

Next day growth will be observed in this plate. Cut the filter paper of size of

agar plate lid and tip in the chloroform.7. Place the filter paper in the cover of nutrient agar and place over the plate.8. Place this plate under Laminar Flow Hood for 30 minutes.9. Now dilute inoculums of indicator strain with normal saline and compare it

with McFarland solution. Now soak cotton bud in these inoculums and

make lawn on nutrient agar plate. Ensure completion of formation of bed

by horizontal, vertical swabbing.

10. Incubate at 37c for 24 hours.11. Check for results next day.


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B. Direct Inoculation Method:

Prepare nutrient agar plate and incubate it. Prepare 100ml nutrient broth 250ml flask and autoclave it. Take fresh indicator and producer strain cultures. Inoculate producer / test strain in nutrient broth with the help of wire loop. Incubate it at 37c for 24 hours. Nutrient broth will become turbid next day, indicating that test strain

produced.

Take sample from nutrient agar flask. Transfer sample to test tube or centrifuge tubes previously sterilized. Centrifuge it for 25-30 minutes at 400 rpm or 2000 rpm for 15 minutes In this test supernatant clear solution is obtained and pellets are present in

the bottom. Pellets are present in the bottom.

Pellets are bacterial mass so we discard this and shift supernatant to anothertest tube.

This supernatant is the antibiotic carrying solution. Now make nutrient brothin tube by placing 2ml solution in tube and then autoclave it.

Transfer the indicator strain to nutrient broth tube. Incubate tube for 24hours at 37c.

Dilute it with 0.9% NaCl solution till turbidity match with macfarland solution. Dip cotton swab in it and form lawn on nutrient agar plate. Make wells in plate with the help of sterile bores incubate plate. Check for results.

Results:

If growth is inhibited and transparent area is obtained this indicator fineresult.

Negative result is indicating by overlapping due to growth.Precautions:

Try to keep two solutions apart to avoid irregular zone.


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Experiment No #3Enzymes Production Ability of Selected Bacterial Species

Enzymes:

These are the secondary metabolites of bacteria produced during themetabolic reaction. Enzymes are protein in nature and are used as catalyst to

speed up the reaction

Introduction:

Enzymes have some impact on the reaction always but never consumed inthe reaction. every cell has about 4000 enzymes in it known as biological

enzymes they take put in metabolic reaction of cell every reaction has a

specific enzyme , because every enzyme has specific sequence of amino acid.

At least 62 amino acids constitute an enzyme , 2500 amino acid sequence

enzymes are also reported known as complex enzymes

Properties of enzymes:Each enzyme possesses certain specific properties for e.g.:

1. Specific in nature i.e. each enzyme catalyzes a specific reaction only.2. Mostly increase the rate of reaction but they are also used for poisoning.3. They are never consumed for take part in the reaction: but always affect the

reaction.

4. They always the designed compounds without chemical contamination.5. Enzymes can also be taken as nutrient supplement

Enzyme production:

Enzymes can be produced by two methods genetic engineering andfermentation. Bacteria produce specific enzymes so are screened for

desired production

Fermentation Method:Fermentation is biological process used to produced or generate a desirable

products under specific condition

Requirements:

Substrate Casein media Petri plate


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ProcedureStep 1: Preparation of nutrient agar plates

Nutrient agar plates are prepared as per composition and number required.incubate for 24 hrs at 37c.Evaluate the sterility

Step2: Freshing of culture

Culture of produce retain is freshed by streaking on nutrient agar plates aincubate.

Step3: Preparation of casein agar plates

Casein plates are prepared as per composition and number required andincubates.

Step4: Transfer of culture

Transfer the fresh culture of producer strain on casein-Agar plate using wireloop making any particular shape then incubate


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Experiment No # 4:Evaluation of enzyme production ability of selected bacterial species at

fermentation level

Fermentation is a biological process used to produced or generate a desirableproduct under specific conditions

Types of fermentation:

Fermentation is usually carried out at 3 levels

small size production pilot plant scale production large scale fermentation

Small size production: It is also known as lab scale or flask scale in which 1 liter or 2 liter flask is

used.

Pilot plant scale production:

It involves trial scale production of desired materialLarge scale fermentation:

It is known as rector scale based or industrial based fermentationEnzymes:

Enzymes are protein molecules composed of certain excipients of aminoacids. Enzymes are composed of 60-2500 amino acid sequence.

Requirements:

Substrate (Casein media) Nutrient agar plates Centrifugation machine Synthetic media Wire loop Nutrient broth Producer strain Autoclave Incubator


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Procedure:Small scale production:

Fresh the producer strain on nutrient agar plates. Prepare 100 ml of nutrientbroth in flask. Prepare 100ml of synthetic media in flask. Autoclave both

flask.

Prepare casein media plates; incubate to evaluate its sterility. Incubate producer strain in 100 ml nutrient broth after introduction. Now introduce 10% inoculums i.e. 10 ml in 90 ml of synthetic media incubate

it at 37 C for 24 hrs. There must be production of enzymes. Centrifuge by

taking sample in centrifuge tube at 10000 rpm for 25-30 mins.

Collect the supernatant at anti sterile plate and discard the pallets. Now evaluate supernatant in sterile plates.

Result:

Milky appearance which conform the presence of enzymes Transparent zone due to hydrolysis of proteins .i.e. complex proteins are

dissolved.


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Experiment No # 5Antibiotic production By Fermentation

Fermentation:

Biological process preceded by the decomposition of certain substancesunder suitable environmental conditions. It may be aerobic or anaerobic e.g.

yogurt or cheese.

Level of fermentation:Three production level for fermentation method

1. small scale production2. Pilot batch trial based fermentation done by RND department from 2-10 l

also called scale up/pilot plant production.

3. Large scale production, rector based on industrial production up to several1000 L.

Optimization of conditions:

Max result of products (derived product)obtained from availabilityresources done on small scale also called standardization.

Physical parameter

Temperature Time of incubation Nutrient medium Aeration tube

Carbon sucrose

Glucose (0.5,1%.....not more tha 5%) increase conc lactic acid,conc acidicdecrease bacterial growth

Nitrogen source

Glycin,glutamic acid,aspartate acidPrimary media

Natural media,growth media,con is unknown,consc of which bacterial growthfacilitated e.g Agar (1.5-2.5) ptimize conc is not knownSecondary media

Synthetic media, production media concentration is known at whichproduction is facilitated optimize concentration is find out at which

production is max .e.g. carbohydrates, proteins; minerals concentration can

be variated to get max product.

Compounds are optimized to get maximum production of antibiotic so calledas production media or synthetic media.


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Example of fermentation

Cheese production Yogurt production Antibiotics Hormones Immounu-modulater

Requirements

Agar plates Wire loops Synthetic media 0.9% Nacl solution Cotton swab Cork

Flask Beakers Burner Graduated cylinder

ProcedureA. Preparation of nutrient agar plates

Nutrient agar plates are prepared As per composition and number required these are inoculated at 37 C

for 24 hour to evaluate sterility

B. Freshening of culture

Using a wire loop streak strains of producer and indicator

Organism on freshly prepared nutrient agarC. Preparation of media

Prepare 100ml of nutrient broth in a flask as nutrient media prepare 90mlof synthetic media in another flask .Prepare three test tubes containing

2ml of nutrient broth each. Incubate these solution at 37 C for 24 hours,

to evaluate sterility

D. Inoculation of producer stain

Inoculate the producer strain in 100ml nutrient broth flask and incubatefor 24 hours. The media becomes turbid due to bacterial growth and

antibioticsE. Inoculation of indicator stains

Indicator strain are inoculated in the test tube containing nutrients brothand inoculated for 24 hours

F. Shifting of inoculam

Shift 10ml of inoculum of producer stain to 90 m of synthetic media.Incubate it .it will turbid due to bacterial growth and antibiotic produced

G. Centrifugation

Centrifuge synthetic media with the antibiotics at 10,000 RPM for 15-20min. Take supernatant in fresh sterile test tube and discard the plate

H. Evaluation Evaluate the antibiotic method produced


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I. Extraction

Extract the supernatant of centrifuge using chromatography techniquesJ. Purification

After during product is purified using chromatography techniquesK Structure determination

Structure of antibiotics is determined using different techniques e.g NMR, IR etc


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Experiment No # 6ISOLATION OF DNA FROM MAMMALIAN CELLS.

THEORY:

DNA is a part of genetic material which carries specific sequence ofnucleotides that code for a particular protein. The sequence of

nucleotide remains same for generation to generation until a

mutation occurs. DNA contains basic hereditary material called genes.

PURPOSE

DNA isolation is done for: Forensic reason, e-g, DNA finger printing. Genetic engineering & genetic recombination for research purposes. Genetic therapy, e-g to evaluate the missing/defective gene.

REQUIREMENTS: Eppendorf tubes 1.5ml Falcon tubes Centrifuge machine DNA concentrator Micropipette Chemical reagents Incubator Solution

CHEMICAL REQUIREMENTS:Solution A:

Tris-EDTA (buffer at pH 7.5) Sucrose 0.32M (precipitation) Magnesium chloride 25m.meter (cell lysis, ppt) Triton X-100 .1% ( se permeability of cells)

Solution B:

Tris-EDTA NaCl 400mm (cell lysis) SDS (increase permeability) Protenase-K-Enzymes (hydrolyze cellular enzyme)

Solution C:

Phenol pH 7.8 bufferSolution D:

Izoamyl Alcohol Chloroform

Solution C & D are used for solvents extraction.

Phenol = 1 Isoamyl alcohol = 1 Chloroform = 25


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Experiment No # 7Determine the antifungal activity of different synthetic compounds

Introductions:

Fungus:

A fungus is a member of a large group of eukaryotic organisms that includesmicroorganisms such as yeasts and molds (British English: moulds), as well as

the more familiar mushrooms. These organisms are classified as a kingdom,

Fungi, that is separate from plants, animals and bacteria. One major

difference is that fungal cells have cell walls that contain chitin, unlike the cell

walls of plants, which contain cellulose. These and other differences show

that the fungi form a single group of related organisms, named the Eumycota

(true fungi or Eumycetes), that share a common ancestor (a monophyletic

group). This fungal group is distinct from the structurally similar

myxomycetes (slime molds) and oomycetes (water molds). The discipline ofbiology devoted to the study of fungi is known as mycology, which is often

regarded as a branch ofbotany, even though genetic studies have shown that

fungi are more closely related to animals than to plants

Drugs:

Many species produce metabolites that are major sources ofpharmacologically active drugs.

Particularly important are the antibiotics, including the penicillins, astructurally related group of-lactam antibiotics that are synthesized from

small peptides. naturally occurring penicillins such as penicillin G (produced by Penicillium

chrysogenum) antibiotics produced by fungi include: ciclosporin, commonly

used as an immunosuppressant during transplant surgery; and fusidic acid.

Widespread use of these antibiotics for the treatment of bacterial diseases,such as tuberculosis, syphilis, leprosy.

Bioremediation:

Certain fungi, in particular "white rot" fungi, can degrade insecticides, herbicides,pentachlorophenol, creosote, coal tars, and heavy fuels and turn them into

carbon dioxide, water, and basic elements.[203]

Fungi have been shown to

biomineralize uranium oxides, suggesting they may have application in thebioremediation of radioactively polluted sites

Procedure:

There are different methods to evaluate the antifungal activity of syntheticcompounds.

Pour plate technique Hyphical extension inhibitory method
http://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Yeasthttp://en.wikipedia.org/wiki/Moldhttp://en.wikipedia.org/wiki/Spelling_differenceshttp://en.wikipedia.org/wiki/Mushroomshttp://en.wikipedia.org/wiki/Kingdom_(biology)http://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Cell_wallhttp://en.wikipedia.org/wiki/Chitinhttp://en.wikipedia.org/wiki/Cellulosehttp://en.wikipedia.org/wiki/Common_ancestorhttp://en.wikipedia.org/wiki/Monophyletichttp://en.wikipedia.org/wiki/Myxomyceteshttp://en.wikipedia.org/wiki/Oomyceteshttp://en.wikipedia.org/wiki/Biologyhttp://en.wikipedia.org/wiki/Mycologyhttp://en.wikipedia.org/wiki/Botanyhttp://en.wikipedia.org/wiki/Pharmacologyhttp://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/%CE%92-lactam_antibiotichttp://en.wikipedia.org/wiki/%CE%92-lactam_antibiotichttp://en.wikipedia.org/wiki/%CE%92-lactam_antibiotichttp://en.wikipedia.org/wiki/Peptidehttp://en.wikipedia.org/wiki/Penicillin_Ghttp://en.wikipedia.org/wiki/Penicillium_chrysogenumhttp://en.wikipedia.org/wiki/Penicillium_chrysogenumhttp://en.wikipedia.org/wiki/Ciclosporinhttp://en.wikipedia.org/wiki/Immunosuppressanthttp://en.wikipedia.org/wiki/Organ_transplanthttp://en.wikipedia.org/wiki/Fusidic_acidhttp://en.wikipedia.org/wiki/Tuberculosishttp://en.wikipedia.org/wiki/Syphilishttp://en.wikipedia.org/wiki/Leprosyhttp://en.wikipedia.org/wiki/Insecticidehttp://en.wikipedia.org/wiki/Herbicidehttp://en.wikipedia.org/wiki/Pentachlorophenolhttp://en.wikipedia.org/wiki/Creosotehttp://en.wikipedia.org/wiki/Coal_tarhttp://en.wikipedia.org/wiki/Carbon_dioxidehttp://en.wikipedia.org/wiki/Fungus#cite_note-pmid15875713-202http://en.wikipedia.org/wiki/Fungus#cite_note-pmid15875713-202http://en.wikipedia.org/wiki/Fungus#cite_note-pmid15875713-202http://en.wikipedia.org/wiki/Biomineralizationhttp://en.wikipedia.org/wiki/Uraniumhttp://en.wikipedia.org/wiki/Oxidehttp://en.wikipedia.org/wiki/Oxidehttp://en.wikipedia.org/wiki/Uraniumhttp://en.wikipedia.org/wiki/Biomineralizationhttp://en.wikipedia.org/wiki/Fungus#cite_note-pmid15875713-202http://en.wikipedia.org/wiki/Carbon_dioxidehttp://en.wikipedia.org/wiki/Coal_tarhttp://en.wikipedia.org/wiki/Creosotehttp://en.wikipedia.org/wiki/Pentachlorophenolhttp://en.wikipedia.org/wiki/Herbicidehttp://en.wikipedia.org/wiki/Insecticidehttp://en.wikipedia.org/wiki/Leprosyhttp://en.wikipedia.org/wiki/Syphilishttp://en.wikipedia.org/wiki/Tuberculosishttp://en.wikipedia.org/wiki/Fusidic_acidhttp://en.wikipedia.org/wiki/Organ_transplanthttp://en.wikipedia.org/wiki/Immunosuppressanthttp://en.wikipedia.org/wiki/Ciclosporinhttp://en.wikipedia.org/wiki/Penicillium_chrysogenumhttp://en.wikipedia.org/wiki/Penicillium_chrysogenumhttp://en.wikipedia.org/wiki/Penicillin_Ghttp://en.wikipedia.org/wiki/Peptidehttp://en.wikipedia.org/wiki/%CE%92-lactam_antibiotichttp://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/Pharmacologyhttp://en.wikipedia.org/wiki/Botanyhttp://en.wikipedia.org/wiki/Mycologyhttp://en.wikipedia.org/wiki/Biologyhttp://en.wikipedia.org/wiki/Oomyceteshttp://en.wikipedia.org/wiki/Myxomyceteshttp://en.wikipedia.org/wiki/Monophyletichttp://en.wikipedia.org/wiki/Common_ancestorhttp://en.wikipedia.org/wiki/Cellulosehttp://en.wikipedia.org/wiki/Chitinhttp://en.wikipedia.org/wiki/Cell_wallhttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Kingdom_(biology)http://en.wikipedia.org/wiki/Mushroomshttp://en.wikipedia.org/wiki/Spelling_differenceshttp://en.wikipedia.org/wiki/Moldhttp://en.wikipedia.org/wiki/Yeasthttp://en.wikipedia.org/wiki/Eukaryote


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Preparation of the crude extracts:

Hundred grams of each of the air-dried and coarsely powdered plant materialwas exhaustively extracted for 2 hours with petroleum ether (60-80

oC) in

soxhlet apparatus.

The petroleum ether extract was filtered and evaporated under reducedpressure using Rota-vapor.

The extracted plant material was then air-dried, repacked in the soxhletapparatus and exhaustively extracted with methanol (98.8%) for 2 hours.

The methanol extract was filtered and evaporated under reduced pressureusing Rota-vapor.

The extracts were dissolved in dimethyl-sulphoxide to make the finalconcentrations which kept in refrigerator till used.

Simultaneously, water extract was prepared by adding (10ml) of boileddistilled water to 5gm of coarsely powdered plants leaves in a beaker on

water bath with occasional stirring for 4 hours.

The aqueous extract was then filtered and rewashed with small volume ofboiled distilled water and added to the filtrate, which were then adjusted to

(5ml) volume and used immediately

Preparation of the tested organisms:

The fungal cultures (Aspergillus niger (ATCC 9763), Candida albicans (ATCC7596) were maintained on sabouraud dextrose agar, incubated at 25C for

4days.

The fungal growth was harvested and washed with sterile normal saline andfinally suspended in (100ml) of sterile normal saline and the suspension was

stored in refrigerator till used.

Preparation of fungal culture:

Sabouraud glucose-agar media. Following composition was used for this purpose: Pepton10g, glucose 20g, Agar 20g, distil water 1000 ml with 5.4 pH. All the contents were mixed and dissolved in distilled water. The solution was autoclaved at 1200C, 15 Lb/sq inch pressure for20 minutes. Pathogens were isolated with the help of sterilized forceps and plated on

sterilized potato dextrose agar (PDA) medium (potato starch: 20 g, dextrose: 20

g, agar: 20 g and distilled water to make the volume 1 liter, which was sterilized

in a gas operated autoclave at 15 pounds pressure per square inch (PSI) for 20

minutes.

Plates were incubated at 25C and observed daily for emergence of colonies. Sub-culturing was done from single spore to obtain pure culture.


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In vitro testing of extracts for antimicrobial activity:

Hyphal extension inhibitory method:

Diffusates were added in Potato dextrose agar (PDA) @ 10, 50, 100 and 200 g L-1and poured into Petri dishes.

PDA medium added only with ethanol and water served as control. Each Petri dish was inoculated with 5 mm plug of pure Isolate taken from

margins of actively growing culture of pathogen.

Then Petri plates were incubated at 25 2oC. Mycelial growth was recorded when the growth of three selected pathogens

were completed in the control treatment. Each treatment was repeated five

times.

Mean radial mycelial growth of each plant Diffusates was recorded and datawere subjected to Statistical analysis.

Radial mycelial growths on different Diffusates were transformed into inhibitionpercentage by using the following formula (Naz et al., 2006):

Inhibition percentage =100 - Mycelial growth on Diffusates Mycelial growth oncontrol X 100


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Pour plate technique:

The cup-plate agar diffusion method (pour plate) was adopted according toKavanagh, (1972) to assess the antifungal activity of the prepared extracts.

One full loop spore of fungus was thoroughly mixed with 60 Ml of SDA. 20 ml of the inoculated SDA were distributed into Sterile Petri dishes. The agar was left to set and in each of these plates 4 cups, 10 mm in Diameter,

was cut using a sterile cork borer No. 4 and the agar discs were removed.

Alternate cups were filled with 0.1ml of each extracts using micro titer-pipetteand Allowed to diffuse at room temperature for two hours.

The plates were then incubated in the upright position at 25C for 4 to 7 days. Two replicates were carried out for each extract against each of the test

organism.

Simultaneously addition of the respective solvents instead of extracts was carriedout as controls.

After incubation the diameters of the results and growth inhibition zones weremeasured, averaged and the mean values were tabulated


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Experiment No # 8Antioxidant activity of different chemical compounds

Antioxidant

An antioxidant is a molecule capable of inhibiting the oxidation of other molecules.

Oxidation is a chemical reaction that transfers electrons from a substance to an

oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals

can start chain reactions that damage cells. Antioxidants terminate these chain

reactions by removing free radical intermediates, and inhibit other oxidation

reactions. They do this by being oxidized themselves, so antioxidants are often

reducing agents such as thiols, ascorbic acid or polyphenols.[1]

Although oxidation reactions are crucial for life, they can also be damaging; hence,

plants and animals maintain complex systems of multiple types of antioxidants, suchas glutathione, vitamin C, and vitamin E as well as enzymes such as catalase,

superoxide dismutase and various peroxidases. Low levels of antioxidants, or

inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill

cells.

As oxidative stress might be an important part of many human diseases, the use of

antioxidants in pharmacology is intensively studied, particularly as treatments for

stroke and neurodegenerative diseases. However, it is unknown whether oxidative

stress is the cause or the consequence of disease.

Antioxidants are widely used as ingredients in dietary supplements in the hope of

maintaining health and preventing diseases such as cancer,coronary heart disease

and even altitude sickness. Although initial studies suggested that antioxidant

supplements might promote health, later large clinical trials did not detect any

benefit and suggested instead that excess supplementation may be harmful.[2][3]

In

addition to these uses of natural antioxidants in medicine, these compounds have

many industrial uses, such as preservatives in food and cosmetics and preventing the

degradation ofrubber and gasoline
http://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Redoxhttp://en.wikipedia.org/wiki/Chemical_reactionhttp://en.wikipedia.org/wiki/Electronhttp://en.wikipedia.org/wiki/Oxidizing_agenthttp://en.wikipedia.org/wiki/Free_radicalhttp://en.wikipedia.org/wiki/Chain_reactionhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Reducing_agenthttp://en.wikipedia.org/wiki/Thiolhttp://en.wikipedia.org/wiki/Ascorbic_acidhttp://en.wikipedia.org/wiki/Polyphenol_antioxidanthttp://en.wikipedia.org/wiki/Antioxidant#cite_note-Sies-0http://en.wikipedia.org/wiki/Antioxidant#cite_note-Sies-0http://en.wikipedia.org/wiki/Antioxidant#cite_note-Sies-0http://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Glutathionehttp://en.wikipedia.org/wiki/Vitamin_Chttp://en.wikipedia.org/wiki/Vitamin_Ehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Catalasehttp://en.wikipedia.org/wiki/Superoxide_dismutasehttp://en.wikipedia.org/wiki/Peroxidasehttp://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Oxidative_stresshttp://en.wikipedia.org/wiki/Pharmacologyhttp://en.wikipedia.org/wiki/Strokehttp://en.wikipedia.org/wiki/Neurodegenerative_diseasehttp://en.wikipedia.org/wiki/Dietary_supplementshttp://en.wikipedia.org/wiki/Cancerhttp://en.wikipedia.org/wiki/Coronary_heart_diseasehttp://en.wikipedia.org/wiki/Altitude_sicknesshttp://en.wikipedia.org/wiki/Clinical_trialhttp://en.wikipedia.org/wiki/Antioxidant#cite_note-1http://en.wikipedia.org/wiki/Antioxidant#cite_note-1http://en.wikipedia.org/wiki/Antioxidant#cite_note-1http://en.wikipedia.org/wiki/Preservativeshttp://en.wikipedia.org/wiki/Rubberhttp://en.wikipedia.org/wiki/Gasolinehttp://en.wikipedia.org/wiki/Gasolinehttp://en.wikipedia.org/wiki/Rubberhttp://en.wikipedia.org/wiki/Preservativeshttp://en.wikipedia.org/wiki/Antioxidant#cite_note-1http://en.wikipedia.org/wiki/Antioxidant#cite_note-1http://en.wikipedia.org/wiki/Clinical_trialhttp://en.wikipedia.org/wiki/Altitude_sicknesshttp://en.wikipedia.org/wiki/Coronary_heart_diseasehttp://en.wikipedia.org/wiki/Cancerhttp://en.wikipedia.org/wiki/Dietary_supplementshttp://en.wikipedia.org/wiki/Neurodegenerative_diseasehttp://en.wikipedia.org/wiki/Strokehttp://en.wikipedia.org/wiki/Pharmacologyhttp://en.wikipedia.org/wiki/Oxidative_stresshttp://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Peroxidasehttp://en.wikipedia.org/wiki/Superoxide_dismutasehttp://en.wikipedia.org/wiki/Catalasehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Vitamin_Ehttp://en.wikipedia.org/wiki/Vitamin_Chttp://en.wikipedia.org/wiki/Glutathionehttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Antioxidant#cite_note-Sies-0http://en.wikipedia.org/wiki/Polyphenol_antioxidanthttp://en.wikipedia.org/wiki/Ascorbic_acidhttp://en.wikipedia.org/wiki/Thiolhttp://en.wikipedia.org/wiki/Reducing_agenthttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Chain_reactionhttp://en.wikipedia.org/wiki/Free_radicalhttp://en.wikipedia.org/wiki/Oxidizing_agenthttp://en.wikipedia.org/wiki/Electronhttp://en.wikipedia.org/wiki/Chemical_reactionhttp://en.wikipedia.org/wiki/Redoxhttp://en.wikipedia.org/wiki/Molecule

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