In The Human Body Oxidation Common Process 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.

Oxidation is a chemical process in which there is addition of oxygen with a loss of electrons or a process in which there is a gain of proton of an atom, molecule or ion.

In the human body oxidation is a common process and occurs billion times in the body such as oxidation of glucose and generation of energy. In this reaction the use of oxygen for energy formation leads to the formation of free radicals or reactive oxygen species (ROS). In this the superoxide anion are produced as a by product of several steps in electron transport chain.

ROS are nothing but the free radicals having unpaired electrons in their outermost orbit, so these free radicals are unstable and to gain stability they try to get electrons from another atom or molecule and thus they make them unstable and results in perilous chain reaction.

As oxidation is common in body and ROS are the output of that reaction and they play important role in cell signalling, so they are important in synthesis of DNA< RNA and proteins and also used in immune system, so these are important in body. But sometime there is an excess production of free radicals in certain conditions like mitochondrial ETC or excess stimulation of NADPH. IN this case body is unable to neutralise them and cause different types of disease such as cancer, atherosclerosis, diabetes, Parkinson's disease, RA, emphysema, malaria, muscular dystrophy etc.

There are following types of free radicals:-

Superoxide ion(O2-):-These are produced from an oxidation reaction having oxygen molecule with an extra electron. Reaction of the superoxide anion with water such as cytosol leads to the formation of hydroxyl radicals. Superoxide ions can harm to DNA, mitochondria etc. As these are the important elements of the body, body save them by neutralising the superoxide ions with the help of enzyme superoxide dismutase

Hydroxyl radical:- These are nothing but the hydroxide ion without any charge. These are highly reactive and cannot be eliminated by enzymatic reaction. These are having very short half-life. These can also damage the proteins, carbohydrates, DNA, lipids and other organic compounds, but these are different from hydroxide ion, this ion is harmless to the body under normal PH.

Singlet oxygen:- This ROS causes oxidation of low density lipoprotein and results in several cardiovascular diseases. Polyphone anti-oxidants can deactivate singlet oxygen.

Hydrogen peroxide:- this is not a free radical but can produce ROS. Body produce enzyme hydrogen peroxidise in self defence to deactivate them.

Perhydroxy radicals:- these are produced from the superoxide radicals and play important role in cell damage by oxidizing allylic carbons.

Antioxidants:- Anti-oxidant are the molecules which inhibit the oxidation of other molecules. These are may be the phytochemicals, vitamins or other nutrient. These are the molecules which protect the cell from free radical and oxidation chain reaction.

Mechanism of anti-oxidation:- Anti-oxidant are the molecules which react with free radicals and neutralise them by oxidising themselves. In this way they end the free radical chain reaction before cell damage.

Antioxidant neutralise the free radical by absorbing the electron and become oxidised, but they get reactivate themselves by passing this electrons to second line elements such as vitamin A, D, E, K.

Factors affecting the effect of antioxidants:-

Nature of free radical

Site of damage

Source of generation of free radical.

Classification of antioxidant:-

There are two types of antioxidants, endogenous and exogenous.

Endogenous antioxidants:-

Superoxide dismutase:- These are one of the most important antioxidant in the body. These are present in different parts of the body like cytoplasm, mitochondria and extracellular fluid. So these antioxidants cover wide range of the body part and can neutralise the superoxide ions.

Catalase:- It's name itself indicate the breakdown of something. This enzyme convert the the hydrogen peroxide into water and oxygen.

Glutathione peroxidise:- these convert the lipidhydrperoxide to their corresponding alcohol and they reduce the hydrogen peroxide.

CoQ10:- this is a mitochondrial co-enzyme it is an essential part of an electron transport chain reaction. It act as an antioxidant and prevent the oxidation of LDL thus helpful in different cardiac diseases.

Hormone antioxidant:-

1)melatonine:- It shows powerful antioxidant activity, so it may be used in the treatment of neoplasm in association with chemotherapy.

Exogenous antioxidant:-

Vitamin antioxidants:-

1) Vitamin A:- It is mainly found in carrots, sweet potatoes, tomatoes, kale, peaches

and apricots. Some of the plant pigments like caretenoids for example lycopene, lutein are act as a precursor of vitamin A.

2)Vitamin C:- It is found in oranges, lime, green pepper, strawberries,

and tomatoes.

3)Vitamin E:- Green leafy vegetables, nuts and seeds, liver and Vegetable oil are the sources of Vitamin C.

Mineral antioxidant:-

Selenium:- Selenium is found to be antioxidant and it shows positive response in normal hamster.

Zinc:- It is found to be helpful in augmentation of antioxidants in high dosage. Study shows that insufficient zinc administration may results in the oxidative stress.

Introduction to rumex sanguineus

General information:-

Rumex sanguineus is a medicinal plant. As a genus rumex there are more than 700 species.

Common name: - Bloody dock, Bloodwort, Red veined dock, Bloody sorrel etc.

Botanical name:- Rumex sanguineus.

Family:- polygonaceae

Biological source:- It is found in California, North America, Washington, it is also widely distributed in Europe. It is also found in southwest asia.

Description:- bloody dock is used as an ornamental plant. It is a perennial plant. It has red veins on its leaflets so it has different common names. It has erect stems containing small star-shaped green flowers which converts into red after sometime then dark brown fruits. It grows in full sun light. Its height is around 1 ft. to 3 ft. It spreads to 6 in. to 12 in. It. It requires well-drained soil. Propagation is done by sowing seeds in situ in spring. It has rosette leaves and tap root.

Chemical constituents:-

Rumex is a medicinal plant. It has lots of uses in herbal medicines. It contains numerous chemical constituents which are helpful in treating lots of diseases

emodin, chrysophanol and physcion are found in the all species of rumex, also some of the species contain aloe-emodin. Mainly it contain anthraquinone glycoside that's why they act as a purgative. It also contain nepodin, physcion, tannin catechol, oxalic acid, oxalate, chrysophanic acid, tannin and complex volatile oil.

Leaves of the rumex contain water, calories, protein, fat, total carbohydrate, fiber, ash, calcium, phosphorus, iron, Vitamin A, thiamine, nicotinic acid, riboflavin, ascorbic acid, rumicin.

USES:-

1) Rumex contain mainly anthraquinone glycoside so it is used in constipation as a purgative in small concentration and laxative in more concentration.

2)It has cholagogue property.

3) In the past it was used in the fungal infection, psoriasis, ringworm and other skin diseases.

3) It has slight anti-bacterial activity for both gram positive and negative bacteria.

4) It contain tannin as a main constituent so it is used in the burn condition and other exudative condition like inflammatory diarrhoea.

5) Decoction of rumex can be used for the treatment of anthrax

6) Root juice of rumex used for toothache.

7) Root decoction of rumex used for the swollen or enlarged lymph nodes. It is also used in the prevention of scurvey, as a depurative agent, also used in the removal of fever.

8) It is also used in the treatment of anaemia, cancer, diarrhoea, eczema, itching, leprosy, rheumatism, syphilis, tuberculosis, urticaria, malaria etc.

9) It is also used in vapour bath to alleviate the eyes and ulcer.

Side effects:- Although it has lots of uses there are some side effects they are a follows

1) In the high concentration it may cause diarrhoea, abdominal cramps, intestinal atrophy and hyphokalemia.

2) As tannin is the main constituent so it has hepatotoxicity.

3) Oxalic acid is found to be one of the toxic acid which form the insoluble calcium salts and thus causes imbalance in calcium concentration and affect the bllod coagulation mechanism and several heart problems.

4) It may cause the nitrate and oxalate poisoning.

5) Tannin is also responsible for the carcinogenicity.

Precautions:-

If the patient is having intestinal obstruction excess use must be avpided.

As it is not a standard stimulant laxative so it should not be used in he constipation during pregnancy.

Anthraquinone can secret into the breast milk and it is present in rumex so it should not be given to breast feeding women.

INSTRUMENTATION

HPLC:-

int_hplc2.jpg (23126 bytes)

Definition:- In the earlier days the liquid chromatography was very slow and gravitation force was used for the separation of organic compounds and it took several days which leads to poor resolution due to diffusion. So to increase the rate of analysis and resolution HPLC system was introduced.

HPLC stands for the high-performance liquid chromatography. In this system pressure is applied to the column with the help of pump which force the mobile phase at high rate through the column and the sample get separated at fast rate.

Principle:- In HPLC separation technique is based on the stationary phase and mobile phase. It may be partition absorption or ion exchange process depending on the type of stationary phase used.

In HPLC compounds get separated depends on their polarity. If the HPLC system contain polar stationary phase and non-polar mobile phase this system is called as normal phase chromatography. In this system separation based on the adsorption to the stationary phase so more non-polar components get eluted first whereas more polar component stick with the stationary phase. If the system is having non-polar stationary phase and polar mobile phase such a system is called as reversed-phase chromatography. In this non-polar component of the samples elute first.

The elution time of a compound depends on the capacity factor(K). This is depends on the chemical nature of analyte, composition and surface area of stationary phase, composition and flow rate of mobile phase and chemical nature of analyte. Column length is also important in the determination of resolution.

Instrumentation for HPLC:-

Solvent release system it contain pump and the filters

Sample inlet system

A column

Detector and recording unit.

Pumps: - These are important because it deliver the solvent or mobile phase in a constant flow rate. There are different types of pumps used HPLC for example pneumatic, syringe, and piston pumps etc. Nowadays more computer controlled metering pumps that can be programmed to vary the ratio of mobile phase as it is required in gradient system. Operating pressure up to 5000 psi with flow rate 10 mL per min is feasible now. Pump components should be made up from inert and resistant to all solvent so that it will not affect in quantitative analysis.

Sample inlet system:- sample inject can be done by manually or there are some auto sample injector to introduce sample at high pressure.

Column:- columns are important part in HPLC as they are the responsible for separation of different components. Columns are mainly made up from the stainless steel so that they can withhold the high pressure. In the normal phase chromatography column contain polar stationary phase and in reverse-phase it contain non-polar stationary phase such as silica.

Detector and recording system:- in the HPLC system sensitivity of the detector should be very high because the quantity of the material applied is very small. Nowadays vast variety of detectors available such as ultraviolet-visible spectrophotometer, fluorimeter, refractive index and mass spectrometer.

System suitability parameters:- these are important because from these parameters we can check the efficacy of the HPLC system. These are as follows

Theoretical plates:- It is believed that each column contain ample of theoretical plates where equilibrium between stationary and mobile phase takes place. As the number of theoretical plates increases efficiency of column also increases. It can be calculated by the equation

N=16(t/w)2

Where, t is the retention time and w is the width at the base of peak.

Retention Time:- It is a time of elution of the peak maximum after Injection of compound

Resolution:- It is the ability of the column to resolve the two solute is of main interest in chromatography. Higher the resolution greater the separation of solute takes place. So it is a function of column efficiency.

Tailing factor:- It is given by the following equation.

W0.05

T= ------------

2f

Where W0.05 is the width of peak at 5% height and f is the distance from peak

maximum to the leading edge of the peak height from the baseline.

Capacity factor:- It is defined as the reduced retention time to the non-retained component.

Chemicals:- Most of the chemicals are obtained from the Fisher Scientific and Sigma Aldrich. Chemicals used for the practical purpose are as follows

2, 2-diphenyl-1-picrylhydrazyl (DPPH), Dulbecco's phosphate buffer saline, ethylenediaminetetracaetic acid (EDTA), Ferric chloride, Ferrous chloride, hydrogen peroxide (H2O2), Thiobarbituric acid (TBA), Trichloroacetic acid (TCA), Rumex sanguineus leave extract was provided by Dr. Soloman Habtemariam (Greenwich University, UK)

METHODOLOGY

HPLC:-

Procedure:- first of mobile phase was prepared using methanol and water in the proportion of 30:70 then it is filtered and degassed in order to remove particulate matter and air bubbles. Sample of rumex leave extract was mixed with methanol in a concentration of 2mg/ml then it is injected with the help of auto injector in a HPLC system having following condition.

Chromatographic condition:-

Isocratic programme

Column

Mobile phase: Methanol and water in the ratio (30:70)

Wavelength: 280 nm and 360 nm

Column temperature: 300C

Flow rate: 0.5ml/min

Run time: 40 min

Injection volume: 20 micro liter

Chromatogram obtained after using mobile phase of methanol and water (30:70) is shown in the fig

Observation:-From the graph it is observed that the chemical components are getting separated but they are not still there is a lot of scope in their separation. So there is need to increase the concentration of methanol so that it can elute more organic compound than water does.

After changing the mobile phase of methanol to water in the proportion of (40:60) obtained chromatogram is shown in the fig

Observation:- from the chromatogram it is observed that more chemical components are separated than the earlier chromatogram.

After changing the mobile phase of methanol to water in the proportion of (50:50) obtained chromatogram is shown in fig

Observation: - from the chromatogram it is observed that there is increase in the number of peaks showing good separation.

To get more separation mobile phase was changed to the proportion of 75% methanol and 25% water and obtained graph was shown in fig.

Observation:- It is observed that separation of the chemical constituents is decreased after 15 min.

Conclusion:- Though there is an increase in the concentration of the methanol it is not separating the chemical constituents properly so there is need to use Gradient system.

Trial 2:- Use of gradient system.

Condition extract of rumex leaves in the concentration of 2 mg/ml in petrol, chloroform and ethyl acetate is analysed by HPLC by using following condition.

Chromatographic condition:-

Gradient programme

Column

Mobile phase: Methanol and water (10:90 to 90:10)

Wavelength: 280 nm and 360 nm

Column temperature: 300C

Flow rate: 0.5ml/min

Run time: 60 min

Injection volume: 20 micro litres.

The analysis of rumex extract by HPLC is shown in the fig.

Observation:- from the chromatogram it is cleared that separation of the organic compounds of the rumex leaves is done appropriately.

DPPH assay:-

Procedure: - DPPH solution of 0.1mM in methanol solution was prepared and then it is incubated in the different concentration of rumex extract in petrol, chloroform and ethyl acetate and two standards of quercetin and rutin which are made in three fold dilutions. All this method is done in 96-well plate microtitre assay. After making the three fold dilutions it is incubated for 20 min. At room temperature and then the resulting solution is analysed with the help of Multiscan EX Reader at a wavelength of 540 nm against blank.

Hydroxyl radical scavenging assay: - All this method is performed in the test tube. First of all mixture of the chemicals containing deoxyribose of 3.75mM, hydrogen peroxide of 1mM, FeCl3 of 100 micro molar, EDTA of 100 micro molar and ascorbic acid of 100 micro molar in Dulbecco's phosphate buffer saline (pH 7.4) was prepared and then the test sample in different solvents such as petrol, chloroform and ethyl acetate and two standard quercetin and rutin in two fold dilutions is mixed in this prepared reaction mixture. last this reaction mixture was incubated for 60 min at 370C. The reaction mixture was ended by adding 1 ml of TBA (1% w/v) and 1 ml of TCA (2% w/v) then heating the tubes in boiling water bath for 15 min. The contents were cooled and transfer to 96-well plate microtitre and then absorbance was measured at 450 nm.

Measurement of reducing power: - 1 ml of reaction mixture was prepared containing different concentration of test compounds made in two fold dilutions in Dulbecco's phosphate buffer saline (pH 7.0) was incubated with potassium ferricyanide (1% w/v) at 500C for 20 min. The reaction was ended by adding TCA solution(10% w/v), ferric chloride (0.1% w/v) in the samples and the absorbance was measured at 700 nm.

RESULTS AND DISSCUSIONS

The chromatogram obtained from the experiment shows that the numbers of chemical components present in the rumex leave extract and there is need of more research for their identification and chemical structure. I look forward for the coming research.

The basic principle behind the DPPH assay is that DPPH is act as a free radical and when it reacts with the antioxidant component it get reduced which is indicated by change in colour and it can be easily monitored by spectrophotometrically.

DILUTION

PETROL 1

2

3

MEAN

% SCAVENGING ACTIVITY

0.27434

0.298

0.331

0.328

0.319

12.84

0.823

0.294

0.337

0.344

0.325

11.2

2.4691

0.311

0.337

0.341

0.329667

10.1

7.4074

0.279

0.278

0.3

0.285667

21.94

22.22

0.293

0.341

0.333

0.322333

12.04

66.66

0.285

0.291

0.291

0.289

21.03

200

0.203

0.187

0.187

0.192333

47.5

CONTROL

0.377

0.364

0.359

0.366667

DILUTION

CHLOROFORM

5

6

MEAN

% SCAVENGING ACTIVITY

0.27434

0.344

0.362

0.314

0.34

1.06

0.823

0.34

0.344

0.353

0.345667

0.58

2.4691

0.33

0.341

0.332

0.334333

2.71

7.4074

0.274

0.276

0.258

0.269333

21.62

22.22

0.271

0.265

0.272

0.269333

21.62

66.66

0.141

0.121

0.118

0.126667

63.142

200

0.051

0.048

0.052

0.050333

85.354

CONTROL

0.335

0.336

0.36

0.343667

DILUTION

ETOAC

8

9

MEAN

% SCAVENGING ACTIVITY

0.27434

0.345

0.319

0.312

0.325333

11.35332

0.823

0.325

0.321

0.333

0.326333

11.08084

2.4691

0.287

0.284

0.297

0.289333

21.12

7.4074

0.179

0.17

0.19

0.179667

51.04

22.22

0.059

0.051

0.069

0.059667

83.74

66.66

0.074

0.047

0.049

0.056667

84.56

200

0.045

0.055

0.046

0.048667

86.73

CONTROL

0.358

0.381

0.362

0.367

DPPH assay for Quercetin

DILUTION

7

8

9

MEAN

% SCAVENGING ACTIVITY

DILUTION

7

8

9

0.06858

0.346

0.339

0.316

0.333667

6.18

0.06858

0.346

0.339

0.316

0.2057

0.332

0.347

0.355

0.344667

3.09

0.2057

0.332

0.347

0.355

0.6172

0.32

0.34

0.327

0.329

7.49

0.6172

0.32

0.34

0.327

1.8518

0.249

0.283

0.253

0.261667

26.42

1.8518

0.249

0.283

0.253

5.55

0.251

0.252

0.228

0.243667

31.49

5.55

0.251

0.252

0.228

DPPH assay for rutin

DILUTION

10

11

12

MEAN

% SCAVENGING ACTIVITY

DILUTION

10

11

12

0.1372

0.338

0.32

0.3

0.319333

3.23

0.1372

0.338

0.32

0.3

0.4114

0.339

0.328

0.323

0.33

6.68

0.4114

0.339

0.328

0.323

1.2344

0.352

0.35

0.33

0.344

11.2

1.2344

0.352

0.35

0.33

3.7033

0.262

0.278

0.273

0.271

12.39

3.7033

0.262

0.278

0.273

After comparing the graphs obtained from the rumex extract leaves in petrol, chloroform and ethyl acetate with the standard antioxidant i.e. querceutin and rutin it is cleared that rumex shows the free radical scavenging activity.

Hydroxyl radicals are one of the potent free radical in the body which are involved in the lots of damaging effects. In the hydroxyl radical scavenging assay autoxidation of ferrous chloride and EDTA complex happens which forms the O2.- which is very unstable and convert to the hydrogen peroxide, this hydrogen peroxide again react with the ferrous chloride to form hydroxyl radical. In the oyer words it is also called as Fenton-type reaction and the reaction shown below

Fe2+ + H2O2 OH. +OH- + Fe3+

Absorbance of samples for hydroxyl radical scavenging assay:-

DILUTIO IN µ gm/ ml

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

250

500

PETROL

0.172

0.182

0.137

0.115

0.096

0.089

0.088

0.12

0.154

0.183

CHCL3

0.17

0.125

0.086

0.108

0.052

0.052

0.047

0.06

0.079

0.102

ETOAC

0.169

0.123

0.121

0.059

0.049

0.044

0.042

0.051

0.053

0.053

Absorbance of standard quercetin

DILUTION IN µ gm/ ml

0.25

0.49

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

Quercetin

0.09

0.078

0.056

0.074

0.053

0.042

0.043

0.043

0.056

0.045

Absorbance of standare rutin

DILUTION IN µ gm/ ml

0.49

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

250

rutin

0.124

0.105

0.085

0.075

0.063

0.053

0.044

0.041

0.045

0.041

Hydroxyl radical Scavenging activity for sample:-

DILUTIO in µ gm/ ml

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

250

500

ETOAC

19.9

38.38

42.65

72.03

76.77

79.14

80.09

75.82

74.88

74.88

CHCL3

19.43

40.75

59.24

48.81

75.35

75.35

77.72

71.56

62.55

51.65

PETROL

18.4

13.74

35.07

45.49

54.5

57.81

58.29

43.12

27.01

13.27

Hydroxyl Radical scavenging activity of Quercetin

DILUTIO in µ gm/ ml

0.25

0.49

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

quercetin

57.34

63.033

73.45

64.92

74.88

80.09

79.62

79.62

73.45

78.67

Hydroxyl Radical scavenging activity of Rutin:-

DILUTIO in µ gm/ ml

0.49

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

250

Rutin

41.23

50.23

59.71

64.45

70.142

74.88

79.14

80.56

78.67

80.56

From the graph it is shown that as the concentration increases hydroxyl radical scavenging activity increases at certain concentration then it remain constant or decreases so it is concluded that the chemical constituent responsible for the hydroxyl radical scavenging is partially soluble in petrol where as in ethyl acetate and chloroform it soluble and shows better activity.

Reducing power is one of the most important properties of anti oxidant. Antioxidants reduce the free radical by donating the electron or proton and get oxidised themselves. In the present study the reductive ability of rumex was assessed on the basis of conversion of ferric to ferrous.

DILUTIO in µ gm/ ml

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

250

500

PETROL

0.128

0.129

0.099

0.144

0.246

0.184

0.281

0.39

0.522

0.875

CHCL3

0.151

0.178

0.175

0.217

0.322

0.448

0.671

0.864

1.109

1.252

ETOAC

0.242

0.234

0.287

0.365

0.587

0.987

1.361

1.93

2.818

2.371

DILUTIO in µ gm/ ml

0.49

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

250

RUTIN

0.116

0.132

0.253

0.182

0.245

0.321

0.611

1.079

1.508

2.318

DILUTIO in µ gm/ ml

0.25

0.49

0.98

1.96

3.91

7.81

15.63

31.25

62.5

125

QUERCE

0.124

0.206

0.197

0.235

0.307

0.599

0.874

1.283

1.765

2.148

From the above readings it is cleared that the rumex can neutralise the free radicals and terminate the free radical chain reaction because the absorbance obtained from the petrol, chloroform and ethyl acetate indicates that it increases as the concentration increases so greater concentration has greater activity and lower one has lower activity.

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.