Ancestors Developed Defense Mechanisms Against Oxygen Toxicity Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

In human life Oxygen is very essential, without oxygen we cannot survive. Our evolutionary ancestors developed defense mechanisms that can minimize the toxic effects of oxygen, without this protection causes the end of life. Natural defences are imperfect, the damage of the cells caused by oxygen can be minimized by using antioxidants. The diseases like cancer, cardiovascular disease, cataracts , age related diseases and degenerative diseases of nervous system. A lot of research works are made in this past decade. In this study the metabolites produced by the oxygen species and with research works they have learned how to prevent the diseases caused by the reactive oxygen species. Now research works are doing for improving the antioxidant activity.

Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and our metabolism. They are continuously produced by body's normal use of oxygen such as respiration and some cell mediated immune functions. The oxygen consumption inherent in cell growth leads to the generation of reactive oxygen species .

Most harmful effects are produced by the reactive oxygen species (ROS) in our body, ROS are act as oxidants. Free radical is a chemical species which have a lone pair of electrons at its outer orbit, so it is unstable and more reactive. Examples are Hydroxyl radical , Nitric oxide radical, Superoxide radical, Lipid peroxyl radical and non radicals are , Hydrogen peroxide , Singlet oxygen, Hypochlorous acid and ozone.

Superoxide radical (O2ï‚·)

Superoxide anion is a reduced form of molecular oxygen created by receiving one electron. Superoxide anion is an initial free radical formed from mitochondrial electron transport systems. Mitochondria generate energy using 4 electron chain reactions, reducing oxygen to water. Some of the electrons escaping from the chain reaction of mitochondria directly react with oxygen and form superoxide anions. The superoxide anion plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide (H2O2), hydroxyl radical (OHï‚·) or singlet oxygen (Oï‚·) in living systems. The superoxide anion can react with nitric oxide (NOï‚·) and form peroxynitrite (ONOOï‚·), which can generate toxic compounds such as hydroxyl radical and nitric dioxide.

Hydrogen peroxide (H2O2)

Hydrogen peroxide is the most stable reactive oxygen species. H2O2 is the primary product of the reduction of oxygen by various oxidase such as xanthine oxidase, uricase, D-amino acid oxidase and -hydroxy acid oxidase localized in peroxisome. Research shows that the H2O2 is the most effective species for cellular injury. The well known Fenton reaction is initiated when Fe2+ comes in contact with H2O2. Ions of Cu, Co, and Ni can also participate in a similar reaction.

Fe2+ + H2O2 Fe3+ + ï‚·OH + OH-

H2O2 also reacts with O2ï‚· to initiate with Haber-Weiss reaction producing ï‚·OH in presence of Fe2+.

O2ï‚· + H2O2 O2 + ï‚·OH + OH-

The most important function of H2O2 is its role as an intracellular signaling molecule .

Hydroxyl radicals (OHï‚·)

Hydroxyl radical is highly reactive. It may react with any molecule present in the cells and hence they are short lived. The life span of OHï‚· at 37ï‚°C is 10-9 sec. The hydroxyl radical is formed from hydrogen peroxide in a reaction catalyzed by metal ions (Fe+ or Cu+), often bound in complex with different proteins or other molecules. This is known as the Fenton reaction.

H2O2 + Cu+/ Fe2+ OH+OH- + Cu+/Fe3+ ………….. (1)

 Superoxide also plays an important role in connection with reaction 1, by recycling the metal ions.

Cu2+ / Fe3+ + O2ï‚· Cu+ / Fe2+ + O2 ..........................(2)

The sum of reaction 1 and 2 is the Haber-Weiss reaction; transition metals thus play an important role in the formation of hydroxyl radicals. Lipid is very sensitive to OHï‚· attack and initiates LPO (Lipid peroxidation). The hydroxyl radical is responsible for DNA damage and LPO.

Nitric oxide (NOï‚·)

Nitric oxide is an inorganic free radical containing odd number of electrons and can form a covalent link with other molecules by sharing a pair of electrons. It is synthesized by an enzyme nitric oxide synthase located in various tissues and plays an active role in free radical tumour biology. NO is synthesized enzymatically from L-arginine by NO synthase

L-arginine + O2 + NADPH ï‚® L-citrulline + NO + NADP+

Nitric oxide is another free radical that has an important biological role. NOï‚· produced in the body relaxes muscles in blood vessels, and lowers blood pressure. But, excess NOï‚· produced in cases of severe infection can be harmful. Unlike HOï‚· or O2ï‚·, NOï‚· is a much slower reacting radical and it combines with other free radical and inhibits further reaction.

Xanthine oxidase (XO)

Xanthine oxidase (XO) is a source of oxygen derived free radicals. XO derives from xanthine dehydrogenase (XD), an initial translational product by proteolysis. In diseased condition, large amount of XO and XD are released into the circulation to produce significant amount of ROMs. XO catalase the oxidation of hypoxanthine to uric acid reducing O2 by one or two electrons resulting in the formation of O2ï‚· or H2O2. During reoxygenation , it reacts with molecular oxygen, thereby releasing superoxide anion radicals, hydrogen peroxide, and further hydroxyl radicals .

Hypoxanthine + O2 ï‚® Xanthine + O2-. + H2O2

Xanthine + O2 ï‚® Uric acid + O2-. + H2O2

Antioxidants

Antioxidants are the substances which scavenges the oxidation process. "Antioxidants are a type of complex compounds found in our diet that act as a protective shield for our body against certain disastrous diseases such as arterial and cardiac diseases, arthritis, cataracts and also premature ageing along with several chronic diseases."

The above definition gives an brief idea about the antioxidants and their function. Recent researches on free radical can made revolution in health and life styles.

Types of antioxidants

Enzymatic and Non-enzymatic.

Antioxidant derived from natural and dietary sources.

Antioxidants based on defense mechanism.

Enzymatic antioxidants

Superoxide dismutase (SOD):- SOD is an endogenously produced intracellular enzyme presents every cell in the body. SOD appears in 3 forms according to the catalytic metal present in the active site.

Glutathione peroxidase (GSH):- It is commonly found in mitochondria and cytosol. Its function is removal of Hydrogen peroxide and organic hydro-peroxide.

Catalase (CAT):- It is found in mitochondria and cytosol. Its main function is the removal of hydrogen peroxide (H2O2).

Non-Enzymatic antioxidants

Carotenoids: It is a lipid soluble antioxidants, commonly seen in membrane tissue. The main function is the removal of reactive oxygen species.

Bilirubin : It is produced by heme metabolism found in blood. The main function is act as extracellular antioxidants.

Glutathione: It is a non protein thiol and found in cells. It has the property of cellular oxidant defense.

Alpha-lipoic acid : It is endogenous thiol. Its property is by serving substitute for glutathione, recycling vitamin C.

Vitamin C : It is found in aqueous phase of cell. Its property is act as free radical scavenger and also act in the recycle of vitamin E.

Vitamin E: It is found in cells. Function is chain breaking antioxidant.

Uric acid: It is a product of purine metabolism. Its property is scavenging of hydroxyl (OH) radical.

Antioxidant derived from natural and dietary sources.

Plants are good source of antioxidants. The leaves of the most medicinal plants have the antioxidant property. The chemical constituents contained in the leaves are causative for the antioxidant property (Eg :- Flavanoids, carotnoids,alkaloids, phenolic alcohols etc.). Daily diet contains full of antioxidants like vegetables, fruits, tea, wine etc.

Secondary products of plants which are functioning as antioxidant are :-

Chlorophyll derivatives.

Essential oils.

Alkaloids.

Carotenoids.

Phytosterols.

Phenolics - coumarines, flavanoids.

Poly phenolics - tannins, proanthocynidine.

Nitrogen containing compounds - alkaloids, indoles.

Antioxidant based on defense mechanism.

Preventive antioxidants : It will suppress the free radical formation eg, enzymes such as peroxidase, catalase, lactoferrin, carotenoids etc.

Radical scavenging antioxidants: It will suppress the chain initiation reaction. Eg, Vitamin C and carotenoids.

Repair and de novo antioxidant: It consist of proteolytic enzymes, it also repairs the DNA of enzymes and genetic materials.

Enzyme inhibitor antioxidants: It induces production and reaction of free radicals and transport of appropriate antioxidants to appropriate active site.

IN VITRO ANTI-OXIDANT STUDIES OF EXTRACTS OF RAW MATERIAL AND ISOLATED COMPOUNDS OF CYPERUS ROTUNDUS LINN RHIZOME

DPPH free radical scavenging activity

Free radical scavenging potentials of HE, CE, EAE and ME of Cr rhizomes and IC1 was tested against a methanolic solution of DPPH. 0.1 mM solution of DPPH in methanol was prepared and 1.0 ml of this solution was added to 3.0 ml of different concentrations (40-200 µg/ml) of PEE, CE, EAE and ME of Cr rhizomes prepared in water. It was incubated at room temperature for 30 minutes and the absorbance was measured at 517 nm against the corresponding blank solution. Ascorbic acid was taken as reference. Percentage inhibition of DPPH free radical was calculated based on the control reading, which contained DPPH and distilled water without any extract.

Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity was measured by the ability of the different fractions to scavenge the hydroxyl radicals generated by the Fe3+-ascorbate-EDTA-H2O2 system. The reaction mixture (final volume of 1.0 ml) contained 100 μl of 2-deoxy2-ribose (28 mM in 20 mM KH2PO4 buffer, pH 7.4), 500 μl of the fractions at various concentrations (50-800 μg/ml) in buffer, 200 μl of 1.04 mM EDTA and 200 μM FeCl3 (1:1v/v), 100 μl of 1.0 mM hydrogen peroxide (H2O2) and 100 μl of 1.0 mM ascorbic acid. The reaction mixture was kept at 37C for 1 h. The free radical damage imposed on the substrate, deoxyribose was measured using the thiobarbituric acid test. One ml of 1% thiobarbituric acid (TBA) and 1.0 ml 2.8% trichloroacetic acid (TCA) were added to the test tubes and the tubes were incubated at 1000C for 20 min. After cooling, the absorbance was measured at 532 nm against a blank containing deoxyribose and buffer. Quercetin (50-800 μg/ml) was used as the positive control.

Hydrogen peroxide scavenging activity

­Hydrogen peroxide solution (2 mM/L) was prepared with standard phosphate buffer (pH 7.4). Different concentration of the fractions (25-400 μg/ml) in distilled water was added to 0.6 ml of hydrogen peroxide solution. Absorbance was determined at 230 nm after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging activity at different concentrations of the fractions was determined and the IC50 values were compared with the standard, α-tocopherol .

Nitric oxide radical scavenging activity

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which were estimated by Griess reagent. The reaction mixture (3 ml) containing 10 mM sodium nitroprusside in phosphate buffered saline, and the fractions or the reference compound (ascorbic acid) at different concentrations (50-800 μg/ml) were incubated at 25C for 150 min. About 0.5 ml aliquots of the incubated sample was removed at 30 min intervals and 0.5 ml Griess reagent was added. The absorbance of the chromophore formed was measured at 546 nm. The inhibition of nitric oxide was measured by comparing the absorbance values of the fractions with that of the standard, ascorbic acid (50-800 μg/ml).

% Scavenging Activity = [(Ac- As) / Ac] Ã- 100

Where, Ac is the absorbance of the control reaction and as is the absorbance in the presence of the sample of the extracts. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in µg/ml) of extracts that inhibits the formation of free radicals by 50%.

Table 21:DPPH SCAVENGING ACTIVITY

Control absorbance [Ac]: 1.9813

Sample

Concentration (µg/ml)

Absorbance at 517 nm [As]

% Anti-oxidant Activity (%)

IC50

(µg/ml)

Petroleum ether

Extract

40

80

120

160

200

1.4588

1.4810

1.4668

1.5813

1.4989

26.3715

25.2510

25.9778

20.1887

24.3476

-

Chloroform Extract

40

80

120

160

200

1.6111

1.5836

1.5518

1.5818

0.9856

18.6847

20.0726

21.6776

20.1635

50.2548

198.49

Ethyl Acetate Extract

40

80

120

160

200

1.6555

1.3221

1.1122

0.9911

0.8691

16.4431

33.2710

43.8651

49.9772

56.1348

169.89

Methanol Extract

40

80

120

160

200

1.3220

1.1889

1.1799

1.1882

1.1642

33.2761

39.9999

40.4481

40.0292

41.2405

-

Standard [ASCORBIC ACID]

40

80

120

160

200

1.4521

1.1114

0.9554

0.8815

0.7591

26.7097

43.9055

51.1779

55.5090

61.6867

115.69

Table 22:% of maximum antioxidant activity of extracts

S. No.

Sample

% maximum Anti-oxidant activity [%]

1

Standard [Ascorbic acid]

61.6867

2

PEE

24.3476

3

CE

50.2458

4

EAE

56.1348

5

ME

41.2405

Table 23: HYDROXY RADICAL SCAVENGING ACTIVITY

Control absorbance [Ac]: 1.0125

Sample

Concentration (µg/ml)

Absorbance at 532 nm [As]

% Anti-oxidant Activity (%)

IC50

(µg/ml)

Petroleum ether

Extract

40

80

120

160

200

1.0015

0.4950

0.9545

0.9666

0.9441

1.0864

1.7283

5.7283

4.5333

6.7555

-

Chloroform Extract

40

80

120

160

200

0.9610

0.9391

0.9329

0.9112

0.9009

5.0864

7.2493

7.8617

10.0049

11.0222

-

Ethyl Acetate Extract

40

80

120

160

200

0.0920

0.8233

0.7144

0.6210

0.4917

10.9135

18.6864

29.4419

38.6666

51.4370

199.89

Methanol Extract

40

80

120

160

200

0.9115

0.8577

0.7277

0.6767

0.5400

9.9753

15.2888

28.1283

33.1654

46.6666

-

Standard [Quercetin]

40

80

120

160

200

0.8992

0.7221

0.6001

0.5111

0.4009

11.1901

28.6814

40.7308

49.5209

60.4049

166.89

Table 24: % of maximum antioxidant activity of extracts

S. No.

Sample

% maximum Anti-oxidant activity [%]

1

Standard [Quercetin]

61.6867

2

PEE

24.3476

3

CE

43.9206

4

EAE

56.1348

5

ME

41.2405

Table 25:HYDROGEN PEROXIDE SCAVENGING ACTIVITY Control absorbance [Ac]: 1.2653

ISOLATED COMPOUND

Concentration (µg/ml)

Absorbance at 230 nm [As]

% Anti-oxidant Activity (%)

IC50

(µg/ml)

Petroleum Ether Extract

40

80

120

160

200

1.1126

1.1022

1.0018

0.9912

0.7814

12.0682

14.7976

20.8251

21.6628

38.2438

-

Chloroform Extract

40

80

120

160

200

1.0008

1.1589

0.9253

0.8112

0.7665

20.9041

8.4090

26.8710

35.8887

39.4214

-

Ethyl Acetate Extract

40

80

120

160

200

0.8992

0.8771

0.7511

0.7119

0.6252

28.9338

30.6804

40.6385

43.7366

50.5887

198.72

Methanol Extract

40

80

120

160

200

0.9100

0.8913

0.8001

0.7112

0.7511

28.0802

29.5582

36.7580

43.6385

40.7919

-

Standard [α-tocopherol]

40

80

120

160

200

0.8662

0.7377

0.6114

0.5514

0.5009

31.5419

41.6976

51.6794

56.4214

60.4125

116.25

Table 26: % of maximum antioxidant activity of extracts

S. No.

Sample

% maximum Anti-oxidant activity [%]

1

Standard [α-tocopherol]

60.4125

2

PEE

38.2438

3

CE

39.4214

4

EAE

50.5887

5

ME

43.6385

Table 27:NITRIC OXIDE SCAVENGING ACTIVITY

Control absorbance [Ac]: 1.0022

Sample

Concentration (µg/ml)

Absorbance at 546 nm [As]

% Anti-oxidant Activity (%)

IC50

(µg/ml)

Petroleum Ether Extract

40

80

120

160

200

0.9980

0.9918

0.9813

0.8119

0.9001

0.4190

1.03771

2.0854

18.9882

10.1875

-

Chloroform Extract

40

80

120

160

200

0.9915

0.9900

0.8331

0.8001

0.8613

1.0676

1.2173

16.6872

20.1656

14.0590

-

Ethyl Acetate Extract

40

80

120

160

200

0.8992

0.7521

0.6219

0.5444

0.4755

10.2773

24.9550

37.9465

45.6795

52.5543

179.97

Methanol Extract

40

80

120

160

200

0.9050

0.8515

0.8133

0.7407

0.6787

9.6986

15.0369

18.8485

26.0925

32.2787

-

Standard [ASCORBIC ACID]

40

80

120

160

200

0.7932

0.6139

0.5311

0.4793

0.3319

20.8541

38.7447

47.0065

52.1752

66.8828

144.98

Table 28: % of maximum antioxidant activity of extracts

S. No.

Sample

% maximum Anti-oxidant activity [%]

1

Standard [Ascorbic acid]

66.8828

2

PEE

18.9882

3

CE

20.1656

4

EAE

52.5543

5

ME

32.2787

Table 29: ISOLATED COMPOUND

ANTIOXIDANT ACTIVITY

Sample

Concentration (µg/ml)

Absorbance at 546 nm [As]

% Anti-oxidant Activity (%)

Dpph scavenging

40

80

120

160

0.9983

0.9810

0.9653

0.9555

0.3891

2.1153

3.6819

10.2354

Hydroxy radical scavenging

40

80

120

160

1.0101

0.9910

0.9833

0.8616

0.2370

2.1234

2.8839

14.9037

Hydrogen peroxide scavenging

40

80

120

160

1.2352

1.2231

1.2122

1.1132

2.3788

3.3351

4.1808

12.0050

Nitric oxide scavenging

40

80

120

160

0.9919

0.9334

0.9119

0.8532

1.0277

6.8648

9.0101

14.8672

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.