In Vitro Studies on The Antioxidant potential of Cyperus rotundus linn.

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In Vitro Studies on

The Antioxidant potential of Cyperus rotundus linn.

1. Introduction

Antioxidants are compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions. There are two basic categories of antioxidants, namely, synthetic and natural. In general, synthetic antioxidants are compounds with phenolic structures of various degrees of alkyl substitution, whereas natural antioxidants can be phenolic compounds (tocopherols, flavonoids, and phenolic acids), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids, and amines), or carotenoids as well as ascorbic acid (Larson, 1988; Hudson, 1990; Hall and Cuppett, 1997). Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used as antioxidants since the beginning of this century. Restrictions on the use of these compounds, however, are being imposed because of their carcinogenicity (Branen, 1975; Ito et al., 1983). Thus, the interest in natural antioxidants has increased considerably (Lo¨liger, 1991). The ability of some phenolic compounds to act as antioxidants has been demonstrated in the literature. Several researchers have investigated the ant oxidative activity of flavonoid compounds and have attempted to define the structural characteristics of flavonoids that contribute to their activity (Nieto et al., 1993; Das and Pereira, 1990; Foti et al., 1996). O-Dihydroxy groups in the B ring, the presence of a C2-3 double bond in conjunction with 4-oxo in the C ring, and 3- and 5-hydroxy groups and the 4-oxo function in the A and C rings are associated with antioxidant activity. Phenolic acids, such as caffeic, chlorogenic, ferulic, sinapic, and pcoumaric acids, appear to be more active antioxidants than the hydroxy derivatives of benzoic acid such as p-hydroxybenzoic, vanillic, and syringic acids (Dziedzic and Hudson, 1983; Larson, 1988). Burton and Ingold (1981) have shown that R-tocopherol is one of the most active in vitro chain-breaking antioxidants. Carotenoids also have a protective function against oxidative damage, and singlet oxygen is very powerfully quenched by â-carotene (Foote et al., 1971). Many of the natural antioxidants, especially flavonoids, exhibit a wide range of biological effects, including antibacterial, antiviral, anti-inflammatory, antiallergic, antithrombotic, and vasodilatory actions (Cook and Sammon, 1996). Antioxidant activity is a fundamental property important for life. Many of the

Biological functions, such as antimutagenicity, ant carcinogenicity, and antiaging, among others, originate from this property (Cook and Samman, 1996; Huang et al., 1992).

The antioxidant activity of several plant materials has recently been reported (Al-Saikhan et al., 1995; Yen and Duh, 1995; Oomah and Mazza, 1996; Wang et al., 1996; Cao et al., 1996; Amarowicz et al., 1996); however, information on the relationship between antioxidant activity and phenolic content and composition of many food plants is not available. The objective of this study

was to determine the contents of total phenolics in several plant products and to explore relationship(s) between phenolic content and antioxidant activity. In

addition, the antioxidant activities of alcoholic extracts of herbal products, such as ginseng, echinacea, and sea buckthorn were compared.

At the very beginning, prior to the footprint of modern civilization, health care system for the mankind was solely dependent on the plant diversity. Curing of diseases and caring of health, natural plants played a vital role all over the universe. Through the history of mankind, naturally occurring plant-derived substances with minimal or no industrial processing have been used to treat illness within local or regional healing practices. This traditional practice is now concerning the promising factor for the development of better health care system and getting significant attention in global health debates.

In the last decade, there has been a global upsurge in the use of traditional medicine(TM) and complementary and alternative medicine (CAM) in both developed and developing countries. Today, therefore, certain forms of traditional, complementary and alternative medicines play an increasingly important role in health care reform globally.

The development of traditional medicines has been influenced by the different cultural and historic conditions in which they were first developed. Their common basic is a holistic approach to life, equilibrium between the mind, body and environment, and an emphasis on health rather than on disease. Generally, the treatment focuses on the overall condition of the individual patient, rather than on the ailment or disease. This more complex approach makes evaluation highly difficult, since so many factors must be taken into account.

Geographical point of view, Bangladesh is enriched with diversified natural plants. Natural plants actually are God gifted asset and with proper utilization mankind can have the chance to develop its whole life through the appropriate collaboration of nature and life.


A medicinal plant is any plant which, in one or more of its organ, contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs. This definition of Medicinal Plant has been formulated by WHO (World Health Organization).

Actually, the plants that possess therapeutic properties or exert beneficial pharmacological effects on the animal body are generally designated as “Medicinal Plants”. Although there r no apparent morphological characteristics in the medicinal plants growing with them, yet they possess some special qualities or virtues that make them medicinally important. It has now been established that the plants which naturally synthesis and accumulate some secondary metabolites, like alkaloids, glycosides, tannins, volatiles oils and contain minerals and vitamins, possess medicinal properties.

Medicinal plants constitute an important natural wealth of a country. They play a significant role in providing primary health care services to rural people. They serve as therapeutic agent as well as important raw materials for the manufacture of traditional and modern medicine. Substantial amount of foreign exchange can be possible to earn by exporting medicinal plants to other countries.


Genesis of modern medicine is actually the continuous innovative process of traditionally utilized plant medicine through its improvement and utilization. Traditional medicine is the synthesis of therapeutic experience of generations of practicing physicians of indigenous system of medicine. Throughout the history of mankind, many infectious diseases have been treated with herbals. The traditional medicine is increasingly solicited through tradipractitioners.

1.3 Table 1:

List of Medicinal Plants and uses

Plant Image Common name Parts Used Medicinal Use
Amla Fruit Vitamin – C,Cough ,Diabetes, Cold,

Laxative, hyperacidity.

Ashok Bark Flower Menstrual Pain,Diabetes,Uterine disorder.
Aswagandha Root, Leafs Restorative tonic, stress, Nerves disorder, Aphrodisiac.
Bael / Bilva Fruit, Bark Diarrhea,Dysentery,Constipation.
Bhumi Amla Whole Plant Anaemic,Jaundice,Dropsy.
Brahmi Whole plant Nervous,Memory enhancer, Mental disorder.
Chiraita Whole Plant Skin Disease,Burning sensation,Fever.
Gudmar / madhunasini Leaves Diabetes,Hydrocil,Asthma.
Guggul Gum rasine Arthritis,Paralysis,Laxative.
Guluchi Stem Gout,Piles,Fever,


Calihari Seed Skin Disease,Labour pain,Abortion, etc.
Kalmegh/ Bhui neem Whole Plant Fever,weekness,Release of gas.
Long peeper / Pippali Fruit, Root Appetizers,Enlarged spleen, Bronchitis, Cold, Antidote.
Makoi Fruit/whole plant Dropsy,Diuretic,Anti dysenteric.
Pashan Bheda Root Kidney stone,Calculus.
Sandal Wood Heart wood , oil Skin disorder,Burning sensation, Jaundice,Cough.
Satavari Root Enhance lactation,General weakness, Fatigue,Cough.
Senna Dry Tubers Rheumatism,Aphrodisiac.
Tulsi Leaves/Seed Cough,Cold,Bronchitis,


Vai Vidanka Root, Fruit, Leaves Skin disease,Snake Bite,Helminthiasis.
Peppermint Leaves, Flower, Oil Digestive,Pain killer.
Gritkumari Leaves Laxative,Wound healing,Skin burns & care,


Sada Bahar Whole Plant Leukemia,Hypotension, Antispasmodic ,Antidote.
Vringraj Seed/whole Anti-inflammatory, Digestive,Hair-tonic.
Swetchitrak Root, Root bar Appetizer,Antibacterial,Anti-cancer.
Rakta Chitrak Root, Root bar Colic,Inflammation,Cough.
Kochila Seed Nervous,Paralysis,Healing wound.
Harida Seed Trifala, wound ulcer, leprosy, inflammation, Cough.
Bahada Seed, Bark Cough, Insomnia, Dropsy, Vomiting, Ulcer, Trifala.
Gokhur Whole Plant Sweet cooling, Aphrodisiac, appetizer, Digestive, Urinary.
Bach Rhizome Sedative, analgesic, antepilepsy, hypertensive.
Vasa Whole Plant Antispasmodic, respiratory, Stimulant.
Nag Champa Bark, Leaf, Flower Asthma, Skin,Burning, Vomiting, Dysentery, Piles.
Benachar Root Burning, ulcer, Skin, Vomiting.
Mandukparni Whole plant Anti-inflammatory, Jaundice, Diuretic, Diarrhoea.
Kaincha/CreeperBaidanka Root, Hair, Seed, Leaf Nervous, Disorder, Constipation, Dropsy.
Dalchini Bark, Oil Bronchitis,Asthma,Cardiac,Disorder,


Kurai Bark, Seed Scabies, Antipyretic, Amoebic dysentery.
Kantakari Whole Plant, Fruit, Seed Diuretic,Anti-inflammatory, Appetizer, Stomachic.

2.1 Practice of Modern medicine

Medicinal plant is pivotal for the development of new drugs. During 1950-1970 approximately 100 plants based new drugs were introduced in USA drug market including deserpidine, reseinnamine, reserpine, vinblastine and vincristine which are derived from higher plants. From 1971-1990 new drugs such as ectoposide, E-guggulsterone, teniposide, nabilone, plaunotol, Z-guggulsterone, lactinan, artemisinin and ginkgo ides were appeared all over the world.2% of drugs were introduced from 1991-1995 including paciltaxel, toptecan, gomishin, irinotecan etc.

Table 2: Chronicle of plant medicine in world market

1950-1970 1971-1990 1991-1995 1996-2008
Deserpidine Ectoposide Paciitaxel Digoxin
Reseinnamine Teniposides Toptecan Taxol
Reserpine E-guggulsterone Gomishin lupeol acetate
Vinblastine Z-guggulsterone Irinotecan Gossypol
Vincristine Nabilone Ginkgolides Hemsleyadin
Serpentine Plaunotol Tubocurarine Qulsqualic acid
Cynarin Lactinan Valapotriates Rescinnamine
Danthron Artemisinin Monocrotaline Yohimbine

Plant based drugs provide outstanding contribution to modern medicine. For example:

Serpentine isolated fromthe root of Indian plant Rauwolfia serpentine in 1953 was a revolutionary event in the treatment of hypertension and lowering of blood pressure.

Vinblastine isolated from Catharanthus rosesus (Farnsworth and Blowster, 1967) is used in the treatment of Hodgkin’s, choriocarcinoma, non-hodgkins lymphomas, leukemia in children, testicular and neck cancer.

Table 3: Some of the important medicinal plants are used for major modern drugs for cancer.

Plant Name Drugs Treatment
Cathranthus rosesus L. (Apocynaceae) Vinblastine and vincristine Hodgkins, Lymphosarcomas and children leukemia.
Podophyllum emodi Wall. (Beriberidaceae) Podophyllotaxin, Testicular cancer, small cell lung cancer and lymphomas.
Taxus brevifolius (Taxaceae) Paciltaxel, taxotere Ovarian cancer, lung cancer and malignant melanoma.
Mappia foetida Miers. Comptothecin, Irenoteccan and Topotecan Lung, ovarian and cervical cancer.
Comptotheca acuminata Quinoline and comptothecin alkaloids used in Japan for the treatment of cervical cancer

Vincristine is recommended for acute lymphocytic leukemia in childhood advanced stages of Hodgkin’s, lymophosarcoma, small cell lung, cervical and breast cancer. (Farnsworth and Bingel, 1977).

Phophyllotoxin is a constituent of Phodophyllum emodi currently used against testicular, small cell lung cancer and lymphomas.

Indian indigenous tree of Nothapodytes nimmoniana(Magpie foetida) are mostly used in Japan for the treatment of cervical cancer.

Plant derived drugs are used to cure mental illness, skin diseases, tuberculosis, diabetes, jaundice, hypertension and cancer. Medicinal plants play an important role in the development of potent therapeutic agents. Plant derived drugs came into use in the modern medicine through the uses of plant material as indigenous cure in folklore or traditional systems of medicine. More than 64 plants have been found to possess significant antibacterial properties; and more than 24 plants have been found to possess antidiabetic properties (Arcamone et al., 1980), antimicrobial studies of plants (Perumal Samy and Ignacimuthu, 1998; 1999 and Perumal Samy et al., 2006), plant for antidotes activity – Daboia russellii and Naja kaouthia venom neutralization by lupeol acetate isolated from the root extract of Indian sarsaparilla Hemidesmus indicus R.Br (Chatterjee, et al., 2006). Which effectively neutralized Daboia russellii venom induced path physiological changes (Alam et al., 1994).

The present investigation explores the isolation and purification of another active compound from the methanolic root extract of Hemidesmus indicus, which was responsible for snake venom neutralization. Antagonism of both viper and cobra venom and antiserum action potentiating, antioxidant property of the active compound was studied in experimental animals. Recently, Chatterjee et al. (2004) from this Nature preceding hdl:10101/npre.2007.1176.1: Posted 28 Sep 2007

Table 4: Plant derived ethno therapeutics and traditional modern medicine

SLNo. Drugs Basic Investigation
1 Codeine, morphin Opium the latex of Papaver somniferum used by ancient Sumarians. Egyptaians and Greeks for the treatment of headaches, arthritis and inducing sleep.
2 Atropine, Hyoscyamine Atropa belladonna, Hyascyamus Niger etc., were important drugs in Babylonian folklore.
3 Ephedrine Crude drug (astringent yellow) derived from Ephedra sinica had been used by Chinese for respiratory ailments since 2700 BC.
4 Quinine Cinchona spp were used by Peruvian Indians for the treatment of fevers
5 Emetine Brazilian Indians and several others South American tribes used root and rhizomes of Cephaelis spp to induce vomiting and cure dysentery.
6 Colchicine Use of Colchicum in the treatment of gout has been known in Europe since 78 AD.
7 Digoxin Digitalis leaves were being used in heart therapy in Europe during the 18th century.

Laboratory reported that an active compound from the Strychnus nux vomica seed extract, inhibited viper venom induced lipid per oxidation in experimental animals. The mechanism of action of the plant derived micromolecules induced venom neutralization need further attention, for the development of plant-derived therapeutic antagonist against snakebite for the community in need. However, the toxicity of plants has known for a long period of time, and the history of these toxic plants side by side with medicinal ones are very old and popular worldwide, they considered the major natural source of folk medication and toxication even after arising of recent chemical synthesis of the active constituents contained by these plants (Adailkan and Gauthaman, 2001; Heinrich, 2000; Pfister et al., 2002).

Teniposide and etoposide isolated from Podophyllum species are used for testicular and lung cancer.

Taxol isolated from Taxus brevifolius is used for the treatment of metastatic ovarian cancer and lung cancer.

The above drugs came into use through the screening study of medicinal plants because they showed fewer side effects, were cost effective and possessed better compatibility.


Medicinal plants are an accessible, affordable and culturally appropriate source of primary health care system in Bangladesh. Marginalized, rural and indigenous people, who can not afford or access formal health care systems, are especially dependent on these culturally familiar, technically simple, financially affordable and generally effective traditional medicines. As such, there is widespread interest in promoting traditional health systems to meet primary health care needs. This is especially true in this country, as prices of modern medicines spiral and governments find it increasingly difficult to meet the cost of pharmaceutical-based health care. Throughout the region, there is strong and sustained public support for the protection and promotion of the cultural and spiritual values of traditional medicines.

The total number of plants with medicinal properties in the subcontinent at present stands at about 2000. About 450 to 500 of such medicinal plants have so far been enlisted as growing or available in Bangladesh.

Table 5: Common name of some Medicinal plants with scientific name and uses found in Bangladesh.

Local name Scientific name Uses
Ulatkambal Abroma augusta Emmenogogue; used in amenorrhoea and dysmenorrhoea.
Muktajhuri Acalypha indica Expectorant, emetic, diuretic; used in bronchitis and asthma.
Apang Achyranthes aspera Purgative, diuretic, ecbolic, hypoglycemic; used in renal dropsy, piles, anasarca, boils and other skin eruptions.
Basak Adhatoda zeylanica Expectorant, bronchodilator, used in cough, asthma, bronchitis, pneumonia, phthisis and respiratory problems.
Bel Aegle marmelos Digestive, stomachic, laxative, astringent; used in constipation and dysentery.
Rashun Allium sativum Carminative, diuretic, hypotensive, used in indigestion, hypertension and diabetes.
Chhatim Alstonia scholaris Febrifuge, ant periodic, astringent, anthelmintic, hypotensive; used in fever, hypertension, diarrhoea and dysentery.
Kalomegh Andrographis paniculata Febrifuge, alterative, stomachic, anthelmintic, cholagogue; used in liver diseases, colic, fever, diarrhea and dyspepsia.
Shatamuli Asparagus racemosus Roots aphrodisiac, alterative, diuretic; promotes lactation; also used in diabetes.
Neem Azadirachta indica Antiseptic; used in fevers, boils, ulcers, eczema and other skin diseases.
Brahmishak Bacopa monniera Blood purifier, brain-, nerve- and cardiac tonic, diuretic; also used for epilepsy.
Nayantara Catharanthus roseus Used in blood cancer, Hodgkin’s disease and diabetes.
Thankuni Centella asiatica Leaf juice is used in cataract and other eye diseases; plant is used in dysentery, internal and external ulcers, and convulsive disorders.
Babchi Cullen corylifolia Seed extract is used in leucoderma, leprosy, psoriasis and inflammatory skin diseases.
Kalo Dhutra Datura metel Narcotic, anodyne and antispasmodic; leaves used in spasmodic asthma, colic, sciatica painful tumours, glandular inflammations.
Ayapan Eupatorium triplinerve Haemostatic and antiseptic; used in ulcers and haemorrhages and also as cardiac stimulant, emetic, diaphoretic and laxative.
Anantamul Hemidesmus indicus Alterative, sudurific, diuretic and blood purifier; used in abdominal tumours.
Kurchi Holarrhena antidysenterica Antidysenteric, astringent, stomachic and anthelmintic. Fruits are hypoglycaemic.
Chalmoogra Hydnocarpus kurzii Seed oil is used as a cure for leprosy and other skin diseases.
Mehedi Lawsonia inermis Paste of leaves and bark is used in burns and scalds, dandruff and various other skin diseases. Decoction in jaundice.
Sarpagondha Raulwolfia serpentina Roots are used as remedy for hypertension, insomnia, anxiety, excitement and insanity.
Ashoke Saraca asoca Strongly astringent and uterine sedative; used in menorrhagia, haemorrhoids and ulcers.
Arjun Terminalia arjuna Bark is hypotensive, cardiac tonic, astringent and febrifuge; has tonic effect on liver cirrhosis.
Methi Trigonella foenum-graecum Diuretic, carminative, emollient, tonic; used in menstrual disorders, diabetes, hypertension and sexual problems
Ashwagondha Withania somniferum Roots are used in headache, convulsions, insomnia, hiccup, coughs and dropsy.
Ada Zingiber officinale Rhizome is carminative, stomachic, digestive; used in dyspepsia, vomiting, loss of voice, coughs, sore throat and fever


Plants have been serving the animal kingdom as its source of energy (food, fuel) as well as its means of shelter and sustenance since the very beginning of its existence on earth’s surface habitable for the animals.

In addition to providing the animal kingdom its food, fuel and shelter, each of these plants has been synthesizing a large variety of chemical substances since their first day of life on earth. These substances include, in addition to the basic metabolites, phenolic compounds, terpenes, steroids, alkaloids, glycosides, tannins, volatile oils, contain minerals, vitamins and a host of other chemical substances referred to as secondary metabolites which are of no apparent importance to the plants own life. But many of these compounds have prominent effects on the animal system and some possess important therapeutic properties which can be and have been utilized in the treatment and cure of human and other animal diseases since time immemorial. These secondary metabolites differ from plant to plant. Thus, the plant kingdom provides a tremendous reservoir of various chemical substances with potential therapeutic properties3.

The chemical constituents, which are capable of influencing the physiological systems of the animal body by exerting some pharmalogical actions, are designated as the active chemical constituents or simply active constituents. In short, it may be said that the chemical constituents present in the medicinal plants constitute the most important aspect of all medicinal plants.

Green leaves are the sites of a great deal of such chemical activity. The chemical substances with medicinal properties found in some plants are the products of such chemical processes. The occurrence of the active chemical substances in all parts of the plant body or they may be accumulated in higher concentrations in some specific part.

The types of chemical constituents are as follows:

A. Alkaloids and amides

· Pyridine group

· Tropane group

· Isoquinoline group

· Quinoline group

· Quinolizidine group

· Indole group

· Steroidal group

· Imidazole group

· Phenylthylamine group

· Alkaloidal amines

B. Antibiotic and Anti-inflammatory principles

C. Bitter and Pungent principles

D. Volatile oils and Fixed oils

E. Glycosides

· Anthraquinone glycosides

· Cardiac glycosides

· Saponin glycosides

· Thiocyanate glycosides

· Other glycosides

F. Gum-resins and Mucilage

G. Vitamins and Minerals.


Like all matter, our bodies are composed entirely of tiny particles called molecules. Each molecule is made up of atoms, and each atom is made up of a center or nucleus and electrons which spin in orbits around it. Ordinarily, the electrons occur in balanced pairs. This keeps the atom and molecule stable. Sometimes a molecule loses one of its electrons or gains and extra one. This causes the molecule to become unbalanced and highly reactive. Such a molecule is called a ‘Free Radical’.

Actually a free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. Many radicals are highly reactive and can either donate an electron to or extract an electron from other molecules, therefore behaving as oxidants or reductants. As a result of this high reactivity, most radicals have a very short half life (10?6 seconds or less) in biological systems, although some species may survive for much longer. The most important free radicals in many disease states are oxygen derivatives, particularly superoxide and the hydroxyl radical.

Free radicals and other reactive oxygen species are derived either from normal essential metabolic processes in the human body or from external sources such as exposure to X-rays, ozone, cigarette smoking, air pollutants and industrial chemicals. Free radical formation occurs continuously in the cells as a consequence of both enzymatic and non-enzymatic reactions. Enzymatic reactions which serve as sources of free radicals include those involved in the respiratory chain, in phagocytosis, in prostaglandin synthesis and in the cytochrome P450 system. Free radicals also arise in non-enzymatic reactions of oxygen with organic compounds as well as those initiated by ionizing radiations.

With electrons unhinged, free radicals roam the body, wreaking havoc. The free radical, in an effort to achieve stability, it takes nearby molecules to obtain another electron and, in doing so, damages those molecules. This situation can be compared to letting a bachelor into a dance where people have come as couples. The bachelor begins cutting in, each time leaving another bachelor, so the breaking up of couples spreads through the dance floor.

If free radicals are not inactivated, their chemical reactivity can damage all cellular macromolecules including proteins, carbohydrates, lipids and nucleic acids. Their destructive effects on proteins may play a role in the causation of cataracts. Free radical damage to DNA is also implicated in the causation of cancer and its effect on LDL cholesterol is very likely responsible for heart disease. In fact, the theory associating free radicals with the aging process has also gained widespread acceptance.


Antioxidants are molecules that can neutralize free radicals by accepting or donating an electron to eliminate the unpaired condition. Typically this means that the antioxidant molecule becomes a free radical in the process of neutralizing a free radical molecule to a non-free-radical molecule. But the antioxidant molecule will usually be a much less reactive free radical than the free radical neutralized. The antioxidant molecule may be very large (allowing it to “dilute” the unpaired electron), it may be readily neutralized by another antioxidant and/or it may have another mechanism for terminating its free radical condition.

A free radical attack on a membrane usually damages a cell to the point that it must be removed by the immune system. If free radical formation and attack are not controlled within the muscle during exercise a large quantity of muscle could easily be damaged. Damaged muscle could in turn inhibit performance by the induction of fatigue. The roles of individual antioxidants have in inhibiting this damage.

The major benefits of Antioxidant through the examination of different health benefits including:

1. Counteraction of the damaging oxidative action of low-density lipoproteins (LDLs), the so-called bad cholesterol, thereby protecting the arteries from worsening effects of atherosclerosis.

2. Protection of the endothelial cells of the arteries themselves from free radical damage, permitting them to be compliant and reactive rather than rigid and dysfunctional.

3. Decrease in platelet aggregation (clumping), protecting the vascular system from clot formation that has potentially damaging effects such as heart attacks and strokes.

4. Counteraction of the oxidation-promoting effects of stress hormones such as the catecholamine’s (epinephrine and nor epinephrine), often secreted in high amounts during chronic stress.

5. Counteraction of free radical damage to many cells of the body that could potentially trigger undesired proliferation in the form of cancer.

6. Protection from some of the damaging effects of aberrant metabolism that can contribute to the triggering of type II diabetes.

7. Protection of important connective tissues of the body to help counteract many age-related forms of deterioration.

8. Protection and enhancement of immune responses important in protective responses against viral infections and surveillance and protection from the formation or spread of many types of cancer.

9. Counteraction of damaging effects of inflammatory responses in diverse systems of the body, including joints (arthritis), and brain (Alzheimer’s disease).

10. Protection against degenerative processes in the brain that can lead to specific neuronal damage associated with Parkinson’s disease and Alzheimer’s disease.

4.1 Classification of anti-oxidant

Antioxidants are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect cell membranes from peroxidation.These compounds may be synthesized in the body or obtained from the diet. The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone mostly present within cells, while others such as uric acid are more evenly distributed. Some antioxidants are only found in a few organisms and these compounds can be important in pathogens and can be virulence factors.

4.1.1 Antioxidant Enzymes:

Even though the production of antioxidant enzymes in the body is a complex process that is not yet totally understood, there are several processes that we are aware of and which seem to constitute a large part of the finished system.

The antioxidant enzyme defense system consists of hundreds of different substances and mechanisms. This is why only an adequate combination of whole foods, such as sprouted food concentrates, will contain all of the known and unknown nutritional factors that the body requires to enhance its antioxidant enzyme supply. Superoxide- Dismutase & Catalase

We are a species that cannot live without breathing. As a result of this circumstance, oxygen has turned into a synonym of life. A fact much less known is that not all oxygen atoms are life supporting. Some are actually quite destructive for our cells. These unhealthy oxygen atoms are unbalanced and constitute the most common “Free Radical” known. The “Oxygen Free Radical” is characterized by having and unpaired electron in its molecular structure. Called “Superoxide”, it is quite capable of causing cell damage.

The first line of defense that the body has against superoxide free radicals is the enzyme known as “Superoxide Dismutase” or (SOD), which is considered the most effective antioxidant. The importance of SOD is so paramount for the protection of our cells, that it represents a substantial proportion of the proteins manufactured by the body. In brief, SOD keeps oxygen under control.

In the process of removing superoxide free radicals, SOD rarely operates alone. It requires the enzyme called “Catalase” or (CAT) to remove hydrogen peroxide molecules which are by-products of the reactions created by SOD. Similar to SOD, CAT is abundant in the body. Integrated in all red blood cells, CAT removes hydrogen peroxide from our tissues, preventing both cell damage and , more importantly, the formation of other more toxic free radicals. In nature, and in the body, SOD and CAT always co-exist.

The natural interactions – synergy – between these two antioxidant enzymes constitute the most effective system of free radical control in our bodies.

Superoxide free radicals initiate the breakdown of “Synovial Fluid” (the lubricating element in the joints of our bodies) causing friction and, eventually, inflammation. For this reason, the attention of clinical SOD research has been focused primarily on inflammatory processes triggered by superoxide free radicals such as arthritis, bursitis and gout.

Deficiency in SOD/CAT is the most notorious nutritional factor in most ‘inflammatory’ processes. Recent applications of SOD/CAT enhancing foods have also proven to be extremely useful as a (pre- & post) operative supplement which stimulates recovery and reduces convalescence periods remarkably.

Considering the powerful link between free radicals and many of today’s health problems, supplements that enhance SOD/CAT activity in the body offer tremendous potential in the field of preventative nutrition. Glutathione Peroxidase

Glutathione Peroxidase is another of the body’s major protectors against free radicals. This antioxidant enzyme consists of the amino acid Glutathione and the trace mineral ‘Selenium’. These two nutrients team up to combat a specific class of free radicals called peroxides. The main biological function of selenium in mammals is a component of the Glutathione Peroxidase enzyme. Many of the attributes of selenium and Glutathione are actually attributes of Glutathione Peroxidase.

Cell membranes consist primarily of lipids (fats). These lipid membranes are very susceptible to damage by free radicals, especially peroxide radicals. This is why rancid fats (lipid peroxides) have proven to be highly toxic. Glutathione Peroxidase prevents destruction of cell membranes by removing several classes of these lipid peroxides.

The main symptoms of excess peroxide free radicals include heart disease, liver disease, premature aging and skin disease such as skin cancer, eczema, wrinkling, age spots, dermatitis and psoriasis. Peroxide free radicals mediate much damage to the body by impairing liver functions. Consisting of nearly 50% fatty tissue, the liver is very susceptible to lipid per oxidative damage.

Although used primarily for skin related problems, many environmentally sensitive and chemically poisoned people report that Glutathione Peroxidase helps them control their allergies and build resistance to the effects of pollution. Generally speaking, all of the antioxidant enzymes are important where pollution is a concern due to their ability to remove free radicals generated by toxic substances. The list of protective effects of Glutathione Peroxidase is growing and is in no way limited to any single symptom such as age spots. The effects of excess per oxidation in our cells are diverse and dangerous and must be limited to maintain cellular health. Methionine Reductase

Methionine Reductase is a unique enzyme that has demonstrated an ability to remove an extremely toxic free radical called the “Hydroxyl Radical”. The hydroxyl radical is commonly formed through reactions involving heavy metals and other less toxic free radicals, such as mercury reacting with hydrogen peroxide. The hydroxyl radical has the ability to damage any type of organic tissue and is considered to be the most dangerous free radical. Hydroxyl radicals are also the main toxins generated by exposure to excessive radiation. With their ability to damage any type of tissue, symptoms directly related to hydroxyl radical induced tissue damage are difficult to identify.

One effective detoxifying application for Methionine Reductase is in the removal of free radical toxins generated by the mercury found in dental fillings. Another interesting application of Methionine Reductase is for the modern day athlete. It seems that hydroxyl radicals are also formed during exercise. This is especially true if we are exercising in oxygen starved closed rooms or in a auto exhaust filled polluted environments. It is amazing to see joggers running along the road during rush hour traffic. Quite possibly, they are doing more harm than good!

Most avid exercisers are always aware of the need to obtain extra nutrition to fuel their activities. What many miss is the importance of cleaning out the extra metabolic wastes that are a direct result of this exercise. Hydroxyl radicals can be created when we burn fat molecules to produce energy as in strenuous exercise or dieting. This is due to the lack of evacuation of chemicals and toxins stored in the fatty tissue which are released when these tissues are used for fuel. These toxins, when not properly evacuated, generate the formation of hydroxyl radicals.

People supplementing their diets with “Methionine Reductase” have reported greater resistance to the ill effects of pollution as well as greater endurance, stamina, flexibility and the ability to recover from extensive exercise. Although it is generally important to exercise, our modern civilized environments force us to compensate for free radical by-products if we wish to gain health or longevity form our workout programs.

4.1.2 The Chain Breaking Antioxidants

Whenever a free radical interacts with another molecule, secondary radicals may be generated that can then react with other targets to produce yet more radical species. The classic example of such a chain reaction is lipid per oxidation and the reaction will continue to propagate until two radicals combine to form a stable product or the radicals are neutralized by a chain breaking antioxidant.

A simplified scheme of free radical initiated lipid per oxidation is summarized here. In the first reaction, hydrogen from an unsaturated fatty acid (RH) is abstracted by a free radical initiator to form a free radical (R·) (I). This is followed by the rearrangement of the double bond and acceptance of oxygen by the free radical to produce a fatty acid peroxyl radical (Roo·) (II). The peroxyl radical then reacts with another molecule of unsaturated fatty acid to form a semis table unsaturated hydro peroxide (ROOH) which also regenerates a molecule of free radical (III). These reactions may propagate unless all the free radical is scavenged by an antioxidant (AH) (IV), or by self-quenching (V).

However, the hydro peroxide may undergo homolytic fission of covalent bonds to form more free radicals (VI, VII). Upon hemolytic fission of the hydro peroxide, a free radical chain reaction may again be initiated. The reaction can be catalyzed via one electron rcdox reaction in the presence of heme or certain heavy metal ions such as copper or iron.

Chain breaking antioxidants are small molecules that can receive an electron from a radical or donate an electron to a radical with the formation of stable byproducts.46 In general, the charge associated with the presence of an unpaired electron becomes dissociated over the scavenger and the resulting product will not readily accept an electron from or donate an electron to another molecule, preventing the further propagation of the chain reaction. Such antioxidants can be conveniently divided into aqueous phase and lipid phase antioxidants. Lipid phase chain breaking antioxidants

These antioxidants scavenge radicals in membranes and lipoprotein particles and are crucial in preventing lipid per oxidation. The most important lipid phase antioxidant is probably vitamin E.47 Vitamin E occurs in nature in eight different forms, which differ greatly in their degree of biological activity. Both classes of compounds are lipid soluble and have pronounced antioxidant properties. They react more rapidly than polyunsaturated fatty acids with peroxyl radicals and hence act to break the chain reaction of lipid per oxidation.

In cell membranes and lipoproteins the essential antioxidant function of vitamin E is to trap peroxyl radicals and to break the chain reaction of lipid peroxidation.53 Vitamin E will not prevent the initial formation of carbon centred radicals in a lipid rich environment, but does minimize the formation of secondary radicals. ?-Tocopherol is the most potent antioxidant of the tocopherols and is also the most abundant in humans. It quickly reacts with a peroxyl radical to form a relatively stable tocopheroxyl radical, with the excess charge associated with the extra electron being dispersed across the chromanol ring. This resonance stabilized radical might subsequently react in one of several ways.

A-Tocopherol might be regenerated by reaction at the aqueous interface with ascorbateor another aqueous phase chain breaking antioxidant, such as reduced glutathione or urate.Alternatively, two A-tocopheroxyl radicals might combine to form a stable dimer, or the radical may be completely oxidized to form tocopherol quinone.

The carotenoids are a group of lipid soluble antioxidants based around an isoprenoid carbon skeleton. The most important of these is ?-carotene, although at least 20 others may be present in membranes and lipoproteins. They are particularly efficient scavengers of singlet oxygen, but can also trap peroxyl radicals at low oxygen pressure with efficiency at least as great as that of A-tocopherol. Because these conditions prevail in many biological tissues, the carotenoids might play a role in preventing in vivo lipid peroxidation.The other important role of certain carotenoids is as precursors of vitamin A (retinol). Vitamin A also has antioxidant properties, which do not, however, show any dependency on oxygen concentration.

Flavonoids are a large group of polyphenolic antioxidants found in many fruits, vegetables, and beverages such as tea and wine. Over 4000 flavonoids have been identified and they are divided into several groups according to their chemical structure, including flavonols (quercetin and kaempherol), flavanols (the catechins), flavones (apigenin), and isoflavones (genistein). There is some evidence that augmenting the intake of flavonoids might improve biochemical indices of oxidative damage.Apart from flavonoids, other dietary phenolic compounds might also make a small contribution to total antioxidant capacity.

Ubiquinol-10, the reduced form of coenzyme Q10, is also an effective lipid soluble chain breaking antioxidant.Although present in lower concentrations than ?-tocopherol, it can scavenge lipid peroxyl radicals with higher efficiency than either ?-tocopherol or the carotenoids, and can also regenerate membrane bound ?-tocopherol from the tocopheryl radical. Indeed, whenever plasma or isolated low density lipoprotein (LDL) cholesterol is exposed to radicals generated in the lipid phase, ubiquinol-10 is the first antioxidant to be consumed, suggesting that it might be of particular importance in preventing the propagation of lipid per oxidation. Aqueous phase chain breaking antioxidants

These antioxidants will directly scavenge radicals present in the aqueous compartment. Qualitatively the most important antioxidant of this type is vitamin C (ascorbate).Ascorbate has been shown to scavenge superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid, aqueous peroxyl radicals, and singlet oxygen. During its antioxidant action, ascorbate undergoes a two electron reduction, initially to the semidehydroascorbyl radical and subsequently to dehydroascorbate.

The semidehydroascorbyl radical is relatively stable owing to dispersion of the charge associated with the presence of a single electron over the three oxygen atoms, and it can be readily detected by electron spin resonance in body fluids in the presence of increased free radical production.

Dehydroascorbate is relatively unstable and hydrolyses readily to diketogulonic acid, which is subsequently broken down to oxalic acid.

Two mechanisms have been described by which dehydroascorbate can be reduced back to ascorbate; one is mediated by the selenoenzyme thioredoxin reductaseand the other is a non-enzyme mediated reaction that uses reduced glutathione. Dehydroascorbate in plasma is probably rapidly taken up by red blood cells before recycling, so that very little, if any, dehydroascorbate is present in plasma.

Uric acid efficiently scavenges radicals, being converted in the process to allantoin A-tocopherol. Urate might be particularly important in providing protection against certain oxidizing agents, such as ozone. Part of the antioxidant effect of urate might be attributable to the formation of stable non-reactive complexes with iron, but it is also a direct free radical scavenger.

Albumin bound bilirubin is also an efficient radical scavenger, and it has been suggested that it might play a particularly crucial role in protecting the neonate from oxidative damage, because deficiency of other chain breaking antioxidants is common in the newborn.

The other major chain breaking antioxidants in plasma are the protein bound thiol groups. The sulphydryl groups present on plasma proteins can function as chain breaking antioxidants by donating an electron to neutralise a free radical, with the resultant formation of a protein thiyl radical. Reduced glutathione (GSH) is a major source of thiol groups in the intracellular compartment but is of little importance in the extracellular space.GSH might function directly as an antioxidant, scavenging a variety of radical species, as well as acting as an essential factor for glutathione peroxidase. Thioredoxin might also function as a key intracellular antioxidant, particularly in redox induced activation of transcription factors

Table 10: Some nutrients & their concentration in the body

Antioxidant metabolite Solubility Concentration in human serum (?M) Concentration in liver tissue (?mol/kg)
Ascorbic acid (vitamin C) Water 50 – 60 260 (human)
Glutathione Water 325 – 650 6,400 (human)
Lipoic acid Water 0.1 – 0.7 4 – 5 (rat)
Uric acid Water 200 – 400 1,600 (human)
Carotenes Lipid ?-carotene: 0.5 – 1, retinol (vitamin A): 1 – 3 5 (human, total carotenoids)
?-Tocopherol (vitamin E) Lipid 10 – 40 50 (human)
Ubiquinol (coenzyme Q) Lipid 5 200 (human) The Transition Metal Binding Proteins

As discussed above, transition metal binding proteins (ferritin, transferring, lactoferrin, and caeruloplasmin) act as a crucial component of the antioxidant defence system by sequestering iron and copper so that they are not available to drive the formation of the hydroxyl radical. The main copper binding protein, caeruloplasmin, might also function as an antioxidant enzyme that can catalyze the oxidation of divalent iron.94

4Fe2+ + O2 + 4H+ ® 4Fe3+ + 2H2O

Fe2+ is the form of iron that drives the Fenton reaction and the rapid oxidation of Fe2+ to the less reactive Fe3+ form is therefore an antioxidant effect.

4.2 Counteracting Free Radical Damage

The body has developed several endogenous antioxidant systems to deal with the production of ROI. These systems can be divided into enzymatic and nonenzymatic groups. Figure summarizes the sites of action of the various antioxidants.

The enzymatic antioxidants include superoxide dismutase (SOD), which catalyses the conversion of O2·-to H2O2 and H2O; catalase, which then converts H2O2 to H2O and O2; and glutathione peroxidase, which reduces H2O2 to H2O.

The nonenzymatic antioxidants include the lipid-soluble vitamins, vitamin E and vitamin A or provitamin a (beta-carotene), and the water-soluble vitamin C and GSH. Vitamin E has been described as the major chain-breaking antioxidant in humans25. Because of its lipid solubility, vitamin E is located within cell membranes, where it interrupts lipid per oxidation and may play a role in modulating intracellular signalling pathways that rely on ROI. Vitamin E can also directly quench ROI, including O2·-, ·OH, and 1O2. The enzymatic and nonenzymatic antioxidant systems are intimately linked to one another and appear to interact with one another.

Both vitamin C and GSH have been implicated in the recycling of alpha-tocopherol radicals. In addition, the trace elements selenium, manganese, copper, and zinc also play important roles as nutritional antioxidant cofactors. Selenium is a cofactor for the enzyme glutathione peroxidase, and manganese, copper, and zinc are cofactors for SOD. Zinc also acts to stabilize the cellular metallothionein pool, which has direct free radical quenching ability. The complex interactions of these different antioxidant systems may imply that therapeutic strategies will depend on combination therapy of various antioxidants rather than a single agent.

Figure 9 describes the interactions among antioxidants. Reactive oxygen intermediates (ROI) induce membrane lipid per oxidation resulting in a chain reaction that can be interrupted by the direct scavenging of lipid peroxyl radicals by vitamin E (VE) and beta-carotene. Vitamin E can then be recycled by both vitamin C (VC) and glutathione (GSH).

The reducing ability of GSH is catalyzed by the enzyme glutathione peroxidase (GSSG). Glutathione is then recycled by NADPH, which is facilitated by glutathione reductase (GSSG). LOO· = active species of the lipid peroxyradical