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In human life Oxygen is really indispensable, without oxygen we can non last. Our evolutionary ascendants developed defence mechanisms that can minimise the toxic effects of O, without this protection causes the terminal of life. Natural defense mechanisms are imperfect, the harm of the cells caused by O can be minimized by utilizing antioxidants. The diseases like malignant neoplastic disease, cardiovascular disease, cataracts, age related diseases and degenerative diseases of nervous system. A batch of research plants are made in this past decennary. In this survey the metabolites produced by the O species and with research plants they have learned how to forestall the diseases caused by the reactive O species. Now research plants are making for bettering the antioxidant activity.

Free groups are cardinal to any biochemical procedure and stand for an indispensable portion of aerophilic life and our metamorphosis. They are continuously produced by organic structure ‘s normal usage of O such as respiration and some cell mediated immune maps. The O ingestion inherent in cell growing leads to the coevals of reactive O species.

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Most harmful effects are produced by the reactive O species ( ROS ) in our organic structure, ROS are act as oxidizers. Free group is a chemical species which have a lone brace of negatrons at its outer orbit, so it is unstable and more reactive. Examples are Hydroxyl extremist, Nitric oxide extremist, Superoxide group, Lipid peroxyl extremist and non groups are, Hydrogen peroxide, Singlet O, Hypochlorous acid and ozone.

Superoxide extremist ( O2i‚· )

Superoxide anion is a decreased signifier of molecular O created by having one negatron. Superoxide anion is an initial free group formed from mitochondrial negatron conveyance systems. Mitochondria generate energy utilizing 4 negatron concatenation reactions, cut downing O to H2O. Some of the negatrons get awaying from the concatenation reaction of chondriosomes straight react with O and signifier superoxide anions. The superoxide anion plays an of import function in the formation of other reactive O species such as H peroxide ( H2O2 ) , hydroxyl extremist ( OHi‚· ) or singlet O ( Oi‚· ) in populating systems. The superoxide anion can respond with azotic oxide ( NOi‚· ) and form peroxynitrite ( ONOOi‚· ) , which can bring forth toxic compounds such as hydroxyl extremist and azotic dioxide.

Hydrogen peroxide ( H2O2 )

Hydrogen peroxide is the most stable reactive O species. H2O2 is the primary merchandise of the decrease of O by assorted oxidase such as xanthine oxidase, uricase, D-amino acid oxidase and i??-hydroxy acid oxidase localized in peroxisome. Research shows that the H2O2 is the most effectual species for cellular hurt. The good known Fenton reaction is initiated when Fe2+ comes in contact with H2O2. Ions of Cu, Co, and Ni can besides take part in a similar reaction.

Fe2+ + H2O2 Fe3+ + i‚·OH + OH-

H2O2 besides reacts with O2i‚· to originate with Haber-Weiss reaction bring forthing i‚·OH in presence of Fe2+ .

O2i‚· + H2O2 O2 + i‚·OH + OH-

The most of import map of H2O2 is its function as an intracellular signaling molecule.

Hydroxyl groups ( OHi‚· )

Hydroxyl extremist is extremely reactive. It may respond with any molecule nowadays in the cells and hence they are short lived. The life span of OHi‚· at 37i‚°C is 10-9 sec. The hydroxyl group is formed from H peroxide in a reaction catalyzed by metal ions ( Fe+ or Cu+ ) , frequently bound in complex with different proteins or other molecules. This is known as the Fenton reaction.

H2O2 + Cu+/ Fe2+ OHi‚·+OH- + Cu+/Fe3+ aˆ¦aˆ¦aˆ¦aˆ¦.. ( 1 )

A Superoxide besides plays an of import function in connexion with reaction 1, by recycling the metal ions.

Cu2+ / Fe3+ + O2i‚· Cu+ / Fe2+ + O2… … … … … … … … .. ( 2 )

The amount of reaction 1 and 2 is the Haber-Weiss reaction ; passage metals therefore play an of import function in the formation of hydroxyl groups. Lipid is really sensitive to OHi‚· onslaught and novices LPO ( Lipid peroxidation ) . The hydroxyl group is responsible for DNA harm and LPO.

Nitric oxide ( NOi‚· )

Nitric oxide is an inorganic free extremist incorporating uneven figure of negatrons and can organize a covalent nexus with other molecules by sharing a brace of negatrons. It is synthesized by an enzyme azotic oxide synthase located in assorted tissues and plays an active function in free extremist tumor biological science. NO is synthesized enzymatically from L-arginine by NO synthase

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

Nitric oxide is another free group that has an of import biological function. NOi‚· produced in the organic structure relaxes musculuss in blood vass, and lowers blood force per unit area. But, extra NOi‚· produced in instances of terrible infection can be harmful. Unlike HOi‚· or O2i‚· , NOi‚· is a much slower responding extremist and it combines with other free extremist and inhibits farther reaction.

Xanthine oxidase ( XO )

Xanthine oxidase ( XO ) is a beginning of O derived free groups. XO derives from xanthine dehydrogenase ( XD ) , an initial translational merchandise by proteolysis. In morbid status, big sum of XO and XD are released into the circulation to bring forth important sum of ROMs. XO catalase the oxidization of hypoxanthine to uric acid cut downing O2 by one or two negatrons ensuing in the formation of O2i‚· or H2O2. During reoxygenation, it reacts with molecular O, thereby let go ofing superoxide anion groups, H peroxide, and farther hydroxyl groups.

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

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

Antioxidants

Antioxidants are the substances which scavenges the oxidization procedure. “ Antioxidants are a type of complex compounds found in our diet that act as a protective shield for our organic structure against certain black diseases such as arterial and cardiac diseases, arthritis, cataracts and besides premature ageing along with several chronic diseases. ”

The above definition gives an brief thought about the antioxidants and their map. Recent researches on free group can do revolution in wellness and life manners.

Types of antioxidants

Enzymatic and Non-enzymatic.

Antioxidant derived from natural and dietetic beginnings.

Antioxidants based on defence mechanism.

Enzymatic antioxidants

Superoxide dismutase ( SOD ) : – Turf is an endogenously produced intracellular enzyme nowadayss every cell in the organic structure. SOD appears in 3 signifiers harmonizing to the catalytic metal nowadays in the active site.

Glutathione peroxidase ( GSH ) : – It is normally found in chondriosome and cytosol. Its map is remotion of Hydrogen peroxide and organic hydro-peroxide.

Catalase ( CAT ) : – It is found in chondriosome and cytosol. Its chief map is the remotion of H peroxide ( H2O2 ) .

Non-Enzymatic antioxidants

Carotenoids: It is a lipid soluble antioxidants, normally seen in membrane tissue. The chief map is the remotion of reactive O species.

Bilirubin: It is produced by haem metamorphosis found in blood. The chief map is act as extracellular antioxidants.

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

Alpha-lipoic acid: It is endogenous thiol. Its belongings is by functioning replacement for glutathione, recycling vitamin C.

Vitamin C: It is found in aqueous stage of cell. Its belongings is act as free extremist scavenger and besides act in the recycle of vitamin E.

Vitamin Tocopherol: It is found in cells. Function is concatenation interrupting antioxidant.

Uric acid: It is a merchandise of purine metamorphosis. Its belongings is scavenging of hydroxyl ( OH ) group.

Antioxidant derived from natural and dietetic beginnings.

Plants are good beginning of antioxidants. The foliages of the most medicative workss have the antioxidant belongings. The chemical components contained in the foliages are causative for the antioxidant belongings ( Eg: – Flavanoids, carotnoids, alkaloids, phenolic intoxicants etc. ) . Daily diet contains full of antioxidants like veggies, fruits, tea, wine etc.

Secondary merchandises of workss which are working as antioxidant are: –

Chlorophyll derived functions.

Essential oils.

Alkaloids.

Carotenoids.

Phytosterols.

Phenolic resins – coumarines, flavanoids.

Poly phenoplasts – tannic acids, proanthocynidine.

Nitrogen incorporating compounds – alkaloids, indoles.

Antioxidant based on defence mechanism.

Preventive antioxidants: It will stamp down the free extremist formation eg, enzymes such as peroxidase, catalase, lactoferrin, carotenoids etc.

Extremist scavenging antioxidants: It will stamp down the concatenation induction reaction. Eg, Vitamin C and carotenoids.

Repair and de novo antioxidant: It consist of proteolytic enzymes, it besides repairs the Deoxyribonucleic acid of enzymes and familial stuffs.

Enzyme inhibitor antioxidants: It induces production and reaction of free groups and conveyance of appropriate antioxidants to allow active site.

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

DPPH free extremist scavenging activity

Free extremist scavenging potencies of HE, CE, EAE and ME of Cr rootstocks and IC1 was tested against a methanolic solution of DPPH. 0.1 mM solution of DPPH in methyl alcohol was prepared and 1.0 milliliter of this solution was added to 3.0 milliliter of different concentrations ( 40-200 Aµg/ml ) of PEE, CE, EAE and ME of Cr rootstocks prepared in H2O. It was incubated at room temperature for 30 proceedingss and the optical density was measured at 517 nanometer against the corresponding clean solution. Ascorbic acid was taken as mention. Percentage suppression of DPPH free group was calculated based on the control reading, which contained DPPH and distilled H2O without any infusion.

Hydroxyl extremist scavenging activity

Hydroxyl extremist scavenging activity was measured by the ability of the different fractions to scavenge the hydroxyl groups generated by the Fe3+-ascorbate-EDTA-H2O2 system. The reaction mixture ( concluding volume of 1.0 milliliters ) contained 100 I?l of 2-deoxy2-ribose ( 28 millimeter in 20 millimeters KH2PO4 buffer, pH 7.4 ) , 500 I?l of the fractions at assorted concentrations ( 50-800 I?g/ml ) in buffer, 200 I?l of 1.04 millimeters EDTA and 200 I?M FeCl3 ( 1:1v/v ) , 100 I?l of 1.0 millimeters H peroxide ( H2O2 ) and 100 I?l of 1.0 millimeters ascorbic acid. The reaction mixture was kept at 37i‚°C for 1 h. The free extremist harm imposed on the substrate, deoxyribose was measured utilizing the thiobarbituric acerb trial. One milliliter of 1 % thiobarbituric acid ( TBA ) and 1.0 milliliters 2.8 % trichloroacetic acid ( TCA ) were added to the trial tubing and the tubings were incubated at 1000C for 20 min. After chilling, the optical density was measured at 532 nanometers against a clean containing deoxyribose and buffer. Quercetin ( 50-800 I?g/ml ) was used as the positive control.

Hydrogen peroxide scavenging activity

A­Hydrogen peroxide solution ( 2 mM/L ) was prepared with standard phosphate buffer ( pH 7.4 ) . Different concentration of the fractions ( 25-400 I?g/ml ) in distilled H2O was added to 0.6 milliliter of H peroxide solution. Absorbance was determined at 230 nanometers after 10 min against a clean solution incorporating phosphate buffer without H peroxide. The per centum scavenging activity at different concentrations of the fractions was determined and the IC50 values were compared with the criterion, I±-tocopherol.

Nitric oxide extremist scavenging activity

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with O to bring forth nitrite ions, which were estimated by Griess reagent. The reaction mixture ( 3 milliliter ) incorporating 10 mM Na nitroprusside in phosphate buffered saline, and the fractions or the mention compound ( ascorbic acid ) at different concentrations ( 50-800 I?g/ml ) were incubated at 25i‚°C for 150 min. About 0.5 ml aliquots of the incubated sample was removed at 30 min intervals and 0.5 milliliter Griess reagent was added. The optical density of the chromophore formed was measured at 546 nanometer. The suppression of azotic oxide was measured by comparing the optical density values of the fractions with that of the criterion, ascorbic acid ( 50-800 I?g/ml ) .

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

Where, Ac is the optical density of the control reaction and as is the optical density in the presence of the sample of the infusions. The antioxidant activity of the infusion was expressed as IC50. The IC50 value was defined as the concentration ( in Aµg/ml ) of infusions that inhibits the formation of free groups by 50 % .

Table 21: DPPH SCAVENGING ACTIVITY

Control optical density [ Ac ] : 1.9813

Sample

Concentration ( Aµg/ml )

Optical density at 517 nm [ As ]

% Anti-oxidant Activity ( % )

IC50

( Aµg/ml )

Petroleum quintessence

Infusion

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 maximal antioxidant activity of infusions

S. No.

Sample

% maximal Anti-oxidant activity [ % ]

1

Standard [ Ascorbic acid ]

61.6867

2

Urine

24.3476

3

Cerium

50.2458

4

EAE

56.1348

5

Maine

41.2405

Table 23: HYDROXY RADICAL SCAVENGING ACTIVITY

Control optical density [ Ac ] : 1.0125

Sample

Concentration ( Aµg/ml )

Optical density at 532 nm [ As ]

% Anti-oxidant Activity ( % )

IC50

( Aµg/ml )

Petroleum quintessence

Infusion

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 maximal antioxidant activity of infusions

S. No.

Sample

% maximal Anti-oxidant activity [ % ]

1

Standard [ Quercetin ]

61.6867

2

Urine

24.3476

3

Cerium

43.9206

4

EAE

56.1348

5

Maine

41.2405

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

ISOLATED COMPOUND

Concentration ( Aµg/ml )

Optical density at 230 nm [ As ]

% Anti-oxidant Activity ( % )

IC50

( Aµg/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 [ I±-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 maximal antioxidant activity of infusions

S. No.

Sample

% maximal Anti-oxidant activity [ % ]

1

Standard [ I±-tocopherol ]

60.4125

2

Urine

38.2438

3

Cerium

39.4214

4

EAE

50.5887

5

Maine

43.6385

Table 27: NITRIC OXIDE SCAVENGING ACTIVITY

Control optical density [ Ac ] : 1.0022

Sample

Concentration ( Aµg/ml )

Optical density at 546 nm [ As ]

% Anti-oxidant Activity ( % )

IC50

( Aµg/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 maximal antioxidant activity of infusions

S. No.

Sample

% maximal Anti-oxidant activity [ % ]

1

Standard [ Ascorbic acid ]

66.8828

2

Urine

18.9882

3

Cerium

20.1656

4

EAE

52.5543

5

Maine

32.2787

Table 29: ISOLATED Compound

ANTIOXIDANT ACTIVITY

Sample

Concentration ( Aµg/ml )

Optical density 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 extremist 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

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