활성산소 Free radical의 개념 - superoxide, H2O2, Hydroxyl radical, lipid hydroperoxide - 논문 읽어야!!

[문형철]

BEYOND REASON

유리기(free radical)

프리라디칼이란?

사과껍질을 벗겨놓은 상태로 방치하면 갈색으로 변함. 이 현상은 사과가 프리라디칼의 공격을 받아 변화되는 결과임. 그러나 사과껍질에 레몬주스 또는 비타민 C를 발라 놓으면 오랫동안 변하지 않음. 비타민  C가 유리기의 공격을 차단시켜주기 때문. 

물질을 이루고 있는 분자는 구조적으로 양자와 그 주위를 둘러싸고 있는 전자로 이루어짐. 전자는 제각기 짝을 이루고 있을때 안정함. 하지만 어떤 원인으로든 짝을 잃으면 분자는 안정상태를 유지하기 위해 다른 전자를 찾음. 이 불안정한 상태의 분자를 프리라디칼이라고 함. 

프리라디칼이 다른 분자로부터 전자를 획득하여 안정상태가 되면 전자를 빼앗긴 다른 분자는 프리 라디칼로 변하여 다른 분자를 공격하게 됨. 인체내에서 이런 반응이 계속된다면 모든 세포가 영향을 받을 수 있음. 

그러나 프리라디칼이 무조건 인체에 해를 입히는 것은 아님. 병원체를 죽인다거나 병든 세포를 죽임으로써 인체를 건강하게 유지시켜주는 역할도 함. 

활성산소(reactive oxygen species, ROS)는 산소(O2)가 4개의 전자를 받으면서 독성이 없는 H2O를 만드는 도중에 생겨나는 부산물이다. 즉, 산소가 전자를 한 개 받으면 superoxide anion(O2-)를 구 성하는데 이 물질은 매우 불안정하여서 효소(superoxide dismutase, SOD)의 작용에 의하여 H2O2(hydrogen peroxide)를 생성한다(그림 1). H2O2는 superoxide anion보다 안정한 물질이어서 세포내에서 대부분은 H2O2을 관찰하게 된다. H2O2는 세포내에서 그 생성이 잘 조절되어서 대부분은 세포내의 항산화 효소인 catalase, peroxidase등에 의하여 독성이 없는 H2O로 제거된다. 그러나, 세포가 조절하지 못할 정도의 많은 양의 H2O2가 생성되고 세포내에서 이를 적절하게 제거하지 못하는 상태에서 H2O2가 전자를 한 개를 더 받으면 OH(hydroxyl radical)이 생성된다. 이 hydroxyl radical이 독성이 매우 강하고 대부분의 세포내의 독성은 이 물질로부터 유래가 된다. 즉, 이 물질이 DNA를 산화하여 DNA 부가물(DNA adduct)을 생성하고 지질(lipid)를 산화하여 lipid peroxide등을 생성함으로써 세포내의 독성을 유발한다. 보통의 경우 활성산소는 superoxide anion, H2O2, hydroxyl radical을 광범위하게 포함하는 것이다.

최근에 많은 연구에 의하면 활성산소(reactive oxygen species, ROS)는 단순히 호흡과정에서 생성되는 부산물이 아니라, 외부자극에 의하여 세포막에 존재하는 수용체를 통하여 일시적으로 생성되는 세포신호전달체계상의 이차신호전달물질(second messenger)로서 작용할 것이라는 연구 결과가 나오고 있다. 즉, 병리적인 상태의 다량의 활성산소는 세포내에서 독성물질로 작용하지만 외부 신호에 의하여 특정 지역에서 일시적으로 생성되는 낮은 농도의 활성산소는 세포의 성장, 사멸과 더불어 면역등의 세포기능을 조절하는 새로운 개념의 세포 내 이차신호전달물질이다 (그림 2 

최근에 이르러서 활성산소종이 세포에 필수적인 이차신호전달물질로 주목받는 이유는 다양한 외부자 극에서 일시적인 생성을 확인하였기 때문이다. PDGF, EGF, bFGF와 같은 펩타이드 성장인자, TGF- β1, IL-1, TNF-α와 같은 분화인자, angiotensin II, thrombin, lysophosphatidic acid와 같은 GPCR의 자극제, Toll-like receptor를 자극하는 자극제를 처리한 다양한 세포에서 활성산소종의 생성이 확인되었으며 생성된 이들을 제거하면 상기에 서술한 자극이 일어나지 않는 것으로 확인되고, 이는 상기의 자극들에서 활성산소가 중요한 매개체 역할을 한다는 것을 증명하는 것이다 (그림 3).

최근 전세계 많은 연구자들은 자극에 의하여 생성된 활성산소가 이차신호전달물질로 작동하기 위해서는 생성체계, 소멸체계(항산화체계)등에 대한 연구를 활발히 진행하고 있다.

활성산소가 많이 생성되는 경우

활성산소가 제거되지 않는 경우

세포내에서 활성산소는 생성과 소멸의 균형에 의하여 일정 소량의 활성산소의 양이 존재하고 이를 통하여 다양한 세포내의 생리적인 현상을 유도한다. 그러나, 생성체계의 과발현 및 소멸체계(항산화체계) 의 돌연변이등을 통한 비정상상태가 유발되면 세포내의 활성산소의 양이 증가되면 다양한 병리현상 (암, 골다공증, 심장질환, 호흡기질환, 치매)을 유발하게 된다  

프리라디칼의 종류

1) Superoxide(O2-)

- 체내에서 에너지를 만들때 미토콘드리아에서 부수적으로 생성되는 프리라디칼

- ATP가 분해되는 과정에서 Hypoxanthine이 xanthine로 변할때 spueroxide가 생김. 

초과산화물 superoxide를 제거하기 위해서는 체내에서 Mn-SOD, Cu/Zn SOD(Superoxide dismutase)가 필요함.

- 미토콘드리아의 기능이 저하됨에 따라 노화현상도 정비례함.

- 구조적으로 미토콘드리아 내막, 외막은 모두 불포화지방산으로 되어 있음. 불포화지방산이 산화되면 과산화지질이라는 강력한 (lipid hydorperoxide)과산화지질이라는 프리라디칼로 변함. 

- 참고로 미토콘드리아가 정상적인 생화학반응을 위해서 필요한 첫번째 요소는 '물"임

2) Hydroxyl radical(OH-) 하이드록실 라디칼

3) hydrogen peroxide(H2O2)

- - 산소가 전자를 한 개 받으면 superoxide anion(O2-)를 구 성하는데 이 물질은 매우 불안정하여서 효소(superoxide dismutase, SOD)의 작용에 의하여 H2O2(hydrogen peroxide)를 생성한다(그림 1). H2O2는 superoxide anion보다 안정한 물질이어서 세포내에서 대부분은 H2O2을 관찰하게 된다. H2O2는 세포내에서 그 생성이 잘 조절되어서 대부분은 세포내의 항산화 효소인 catalase, peroxidase등에 의하여 독성이 없는 H2O로 제거된다. 그러나, 세포가 조절하지 못할 정도의 많은 양의 H2O2가 생성되고 세포내에서 이를 적절하게 제거하지 못하는 상태에서 H2O2가 전자를 한 개를 더 받으면 OH(hydroxyl radical)이 생성된다

4) Lipid hydroperoxide(과산화지질)

- 우리가 섭취하는 음식중 기름이 있는데, 액체상태를 기름, 고체상태를 지방이라고 함. 

- 식물성 기름은 탄소의 수가 18개 이상이며 이중결합을 가짐. 이를 불포화되었다고 표현하고 불포화지방산이라고 함.

- 불포화지방산이 산화될때 즉 식물성 기름이 산화되면 과산화지질이라는 프리라디칼이 됨.

- 인체는 60조개의 세포가 있고 각 세포막은 인지질이라는 불포화지방산으로 되어 있음. 

  활성산소가 세포막의 지방산을 공격할때 과산화지질이 생김. 

  지질과산화작용이 진행되는 동안 지방분자는 무수한 프리라디칼을 방출함. 

  냉동실 고기를 꺼내 상온에 방치할때 상하는 과정과 같은 것임. 

- 과산화지질은 음식으로 섭취될 수도 있고 체내에서 프리라디칼의 공격을 받아 생성될 수도 있음

- 체내에서 과산화지질이 단백질과 결합하면 lipofuscin이라는 물질을 생성시킴. 

  lipofuscin이 피부로 침착되면 검버섯으로 나타나고 뇌로 침착하면 치매가 됨. 

-  따라서 피부에 검버섯이 많다는 것은 노화현상의 표현인 동시에 과산화지질의 섭취가 많고 프리라디칼이 증가되었다고 판단함. 

항산화제(클릭클릭)

- SOD, 비타민 A, C, E, 글루타치온, 코엔자임 큐텐, 알파 lipoic acid, 시스테인, 깅코, 셀레늄 등

오랫동안 찾고 싶었던 내용이 담긴 논문

Review Article
Free Radicals: Implications in Etiology of Chronic Diseases and Their Amelioration through Nutraceuticals

Sadaf Kalam 
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. C.R. Rao Road, Gachibowli, Hyderabad, India

Mir Zahoor Gul 
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. C.R. Rao Road, Gachibowli, Hyderabad, India

Rupal Singh 
Biochemical Research Laboratory, Centre for Scientific Research and Development, People�s University, Bhanpur, Bhopal, M.P. India

Sravani Ankati 
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. C.R. Rao Road, Gachibowli, Hyderabad, India

ABSTRACT:

Background: Free radicals are highly reactive molecular species with an unpaired electron in the outer valence orbitals which causes them to capture electrons from other substances in order to neutralize themselves. As a by-product of oxidative biochemical reactions in the cells, these chemical moieties get commonly produced. During the past several years, there has been a mounting concern in the medical implications of free radicals. They can adversely alter many crucial biological molecules leading to loss of form and function. The association between free radicals, antioxidants and functioning of various organs and organ systems is highly complex. Free radicals induce oxidative/nitrosative stress leading to cellular redox imbalance inducing pathogenesis of several diseases. Free radical damage effectively contributes to the etiology of several chronic health problems such as cardiovascular disease, diabetes, cancer, neurodegenerative disorders and other chronic conditions. Antioxidants are a broad group of nutritional elements which constitute the first line of defense against free radical damage. Nutraceuticals and phytochemicals thus represent an important natural source of antioxidative therapies apart from endogenous mechanisms. 

Objective: The present study highlights generation of free radicals, role of oxidative stress in pathogenesis of chronic diseases and the protective action of antioxidants. The putative role of nutraceuticals/phytonutrients as antioxidative therapeutic agents is also discussed. 

이 논문은 프리라디칼의 생성, 만성질환에서 산화스트레의 역할, 항산화물질의 방어작용에 대해 연구함. 

항산화물질의 효과가 추정되는 분야에 대해서도 논의함. 

Conclusion: Research indicates that functional food is the chief source of mental and physical well-being, thus contributing to prevention and reduction of risk factors for several diseases. Thus, the search has been strengthened for exploring effective, nontoxic natural phytocompounds with antioxidative activity in addition to endogenous antioxidant defense systems to combat the dreadful effects of free radicals and their counterparts.

How to Cite:
Sadaf Kalam, Mir Zahoor Gul, Rupal Singh  and Sravani Ankati , 2015. Free Radicals: Implications in Etiology of Chronic Diseases and Their Amelioration through Nutraceuticals. Pharmacologia, 6: 11-20
DOI: 10..5567/pharmacologia.2015.11.20

INTRODUCTION
Nutrition plays an essential and fundamental role in maintaining overall health of an individual. Poor nutrition tends to have a considerable impact in implication of chronic diseases. Greek physician Hippocrates back in 400 B.C., said, "Let food be thy medicine and medicine be thy food". At least four of the 10 leading causes of death in today’s world like heart disease, cancer, stroke and diabetes can be related to dietary habits. During past several years, medical research indicates a significant relationship between free radicals and antioxidants followed by the implications of these unstable entities in several chronic ailments (Aruoma, 1994; Rao et al., 2006; Lobo et al., 2010). It is paradoxical that oxygen which is an element indispensable for life, can have severely deleterious effects on the human body under certain situations. Nutraceutical word is the amalgamation of "nutrition" and "pharmaceutical" and was coined in 1989 by Stephen DeFelice. Nutraceuticals have been considered to be alternative medicines for the past few years (Kaur et al., 2012).

FREE RADICALS
Free radicals are highly reactive molecular species with an unpaired characterization of natural antioxidant having less or no electrons in the outer orbital. Free radicals that are normal metabolites in aerobic biological systems have varied reactivities, ranging from the high reactivity of hydroxyl radical (t1/2 = 1 nsec) to the low reactivity of melanins (t1/2 = days), with the intermediate reactivity of nitric oxide (t1/2 = 1-10 sec) and ubisemiquinone (t1/2 = 10 msec). Estimates indicate that the average person has around 10000-20000 free radicals attacking each body cell each day (Valdez et al., 2000; Valko et al., 2001). Targets of free radicals include all kinds of biologically relevant molecules in the body including lipids, nucleic acids and proteins, leading to cell damage and homeostatic disruption.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS): Oxygen molecules are pioneers for radical reactions. When O₂gains 1 electron, it turns into a superoxide anion radical, O₂‾. The addition of a second electron (together with 2 protons) turns the latter into hydrogen peroxide, H₂O₂. Peroxide is not a radical but it easily gains a third electron giving rise to a hydroxyl radical, OH^• and hydroxyl ion, HO‾. Hydroxyl radical-OH^•, a very active chemical particle, easily initiates uncontrollable chain reactions. Free radical chain reactions gravely damage important biomolecules in vitro, causing oxidative damage to DNA, proteins and other macromolecules. In biochemical literature, ROS are traditionally considered as highly dangerous particles (Fridovich, 1998). The hydroxyl radical (OH^•) which is the neutral form of hydroxide ion, has a high reactivity, making it very dangerous radical with a very short in vivo half-life of approximately 10^-9 sec. The term Reactive Oxygen Species (ROS), often used in the biomedical free radical literature has been a collective term that includes not only oxygen-centered radicals such as O₂^•‾ and OH^• but also some nonradical derivatives of oxygen, such as hydrogen peroxide (H₂O₂), singlet oxygen ¹O₂ and hypochlorous acid (HOCl). A similar term, Reactive Nitrogen Species (RNS), is also becoming widely used. Some of these species are much less ‘reactive’ than others, e.g., O₂^•‾ and NO^• react directly with less number of molecules in the human body, in comparison to OH^•. When generated in vivo, OH^• will react at its site of formation (Halliwell, 2006). Thus, ROS and RNS formed in vivo are considered to be highly reactive and potentially damaging transient chemical species. These are continuously produced during the body’s metabolic reactions in small amount, as they are essential for energy supply, detoxification, chemical signaling and immune function (Gutowski and Kowalczyk, 2013).

The ROS are regulated by endogenous enzymes superoxide dismutase, glutathione peroxidase and catalase but due to overproduction of reactive species, induced by exposure to external oxidant substances or a failure in the defense mechanism, damage to cell structure, DNA, lipids and proteins occur which increases the risk for more than 30 different disease processes viz., muscle atrophy, atherosclerosis, multiple sclerosis, autoimmune disease, emphysema and cataract to name a few (Percival, 1998).

Free radicals, oxidative stress and antioxidants share an intricate relationship in maintaining health, aging and age-related diseases (Fig. 1). Prooxidants are chemicals which act by triggering oxidative stress by increasing free radicals leading to cell damage or through ROS production or inhibiting antioxidant systems. Oxidative stress is the shift towards the pro-oxidants in pro-oxidant: Antioxidant balance that can occur due to increase in oxidative metabolism. Thus, oxidative stress, defined as ‘the imbalance between oxidants and antioxidants in favor of the oxidants potentially leading to damage’’ has been suggested to be the cause of aging and various disease in humans and is believed to be a critical concept in maintaining a healthy biological system (Kalam et al., 2012). Oxidative stress induced by free radical is counter-balanced by the body’s endogenous antioxidant systems (Wong et al., 2000). "Antioxidants and free radicals exhibit ‘yin-yang relationship’ as propounded in more than 2000 years old traditional Chinese medicine system."

Figure 1: Intricate relationship between free radicals, oxidative stress and antioxidants

An imbalance between the generation of free radicals and antioxidants, including some co-factors may lead to implications for aging, diabetes, cardiovascular disease, cancer, neurodegenerative disorders and other chronic conditions.

Pathways and sources leading to generation of free radicals: Free radicals/ROS/RNS are produced mainly from different sources in the biological system i.e., cellular metabolism like mitochondrial electron transport chain, endoplasmic reticulum oxidation, NADPH oxidase, xanthine oxidase, prostaglandin synthesis, reduced riboflavin, nitric oxide synthetase, reperfusion injury, cytochrome P450, activated neutrophils and phagocytic cells and environmental sources like drugs, pesticides, transition metals, tobacco smoke, alcohol, high temperature, redox cycling of xenobiotics, exposure to physicochemical agents like ionizing radiations such as X-rays and γ-rays besides visible light or UV in the presence of oxygen and an endogenous compound or a drug that act as photosensitizer (Sen et al., 2010). The generation of ROS and the accumulation of mitochondrial DNA (mt DNA) damage, are the common underlying pathophysiological mechanism in many diseases caused by mitochondrial dysfunction (Pieczenik and Neustadt, 2007).

ROS stimulated oxidative damage in biomolecules: ROS, depending upon their nature, can react with major biomolecules such as lipid, protein and DNA, producing different types of secondary radicals like lipid radicals, sugar and base derived radicals, amino acid radicals and thiyl radicals. These radicals in the presence of oxygen are converted to peroxyl radicals. Peroxyl radicals tend to initiate, chain reactions in biological systems. The highest concentration of unsaturated fatty acids is present in lipid components of cellular membranes thus making them vulnerable to the oxidation by ROS. The ROS react with membrane lipids causing lipid peroxidation, resulting in the formation of lipid hydroperoxide (LOOH) which can further de-compose to an aldehyde such as malonaldehyde, 4-hydroxy nonenal (4-HNE) or form cyclic endoperoxide, isoprotans and hydrocarbons. The consequences of lipid peroxidation are cross linking of membrane proteins, change in membrane fluidity and the formation of lipid-protein, lipid-DNA adduct which may be detrimental to the functioning of the cell. ROS possess the potential to interact with proteins directly and indirectly both, resulting into peroxidation, changes in their tertiary structure, proteolytic degradation, protein-protein cross linkages and fragmentation. Free radicals such as -OH^• can fragment and crosslink proteins. The side chains of all amino acid residues of proteins, in particular tryptophan, cysteine and methionine residues are susceptible to oxidation by ROS. Carbonyls such as aldehydes and ketones are normal protein oxidation products (Bent and Hayon, 1975). During the oxidation of aromatic residues, the formation of phenoxyl radicals from tyrosine and their conversion into dityrosine and further products can occur, especially if there are no reductants to repair the tyrosil radicals (e.g., thiols, vitamin E) and if there are vicinal tyrosil radicals. Histidine in reactions with free radicals can form some imidazole decay products or in some cases, aspartic acid and can also form some histidine derivatives (Dean et al., 1997).

DNA is a stable, well-protected molecule but ROS can interact with it and cause several types of damage including modification of DNA bases, single and double strand DNA breaks loss of purines, damage to the deoxyribose sugar, DNA-protein cross-linkage and damage to the DNA repair system (Beckman and Ames, 1997). The OH^• radical reacts with DNA producing a variety of adducts. It can attack purine and pyrimidine bases to form OH^• radical adducts which are both oxidizing and reducing in nature. This induces base modifications and sometimes release of bases. Some of the important base modifications include 8-hydroxydeoxyguanosine (8-OHdG), 8 (or 4-,5-)-hydroxyadenine, thymine peroxide, thymine glycols and 5-(hydroxymethyl) uracyl5. Free radicals can also attack the sugar moiety which can produce sugar peroxyl radicals and subsequently inducing strand breakage. The consequence of DNA damage is the modification of genetic material resulting into cell death, mutagenesis, carcinogenesis and aging (Kunwar and Priyadarsini, 2011). All ROS are not capable of damaging DNA but OH^•, radicals are the major source of endogenous DNA damage in aerobic organisms (Marnett, 2000). Oxygen-free-radical damage nucleic acids resulting in production of many different types of lesions which can be grouped into strand breaks and base modification products (Cooke et al., 2003). Superoxide produced from the xanthine/xanthine oxidase system has been shown to cause DNA strand breaks. Hydroxyl free radicals react readily with nucleic acids, yielding many different kinds of products including strand breaks which are probably the most frequent single lesion.

ANTIOXIDANTS
‘Antioxidants’ are substances that neutralize free radicals or their actions (Sies, 1996). Being protective agents, they are capable of stabilizing or deactivating free radicals before they attack cells. Being beneficial compounds, they control free radical formation naturally and help organisms to deal with oxidative stress caused by free radicals. Exposure to free radicals necessitates intake of antioxidant supplemented diets to shield cells from the adverse effects of free radicals. A free radical is referred to as-"Electron thief" and antioxidant as "electron donor" (Fig. 2). The antioxidant offers the free radical the extra electron, so the free radical does not have to steal one.

The body relies on several endogenous defense mechanisms to help protect against free radical-induced damage (Kumar et al., 2008; Rahman, 2007; Adachi et al., 1994). There is currently considerable interest in dietary antioxidants as bioactive components of food. Identification of pharmacologically potential antioxidant compounds has increased tremendously as they exhibit no side effects for use in preventive medicine and food industry. Antioxidant compounds are present in vegetables, fruits and many natural beverages like tea. Balanced diets are naturally rich in antioxidants. Antioxidants display an array of benefits like they support kidney function, maintain good dental health, improve reproductive and nervous system functioning have anti-aging effect, protect liver, support immune system and improve defense power of the body. They also reduce obesity, offer protection against digestive disorders, maintain healthy vision and improve quality of sleep.

Nonspecific antioxidant molecules: Due to the generation of highly reactive oxidants, such as hydroxyl radical OH^•, mammalian cell is constantly undergoing wear and tear causing grave damage to cell which has to be restricted. Thus, a second line of defense against free radical attack is constituted by nonenzymatic antioxidant molecules, such as vitamin E, vitamin C, ubiquinone or Coenzyme Q and carotenoids. Reduced glutathione might act as a cellular antioxidant while α-tocopherols might function as chain-breaking antioxidants because they effectively interrupt free radical propagation reactions (Paravicini and Touyz, 2008).

Free radicals induced human pathologies: Free radicals induce oxidative stress which has been implicated in the etiology of myriad of chronic diseases including atherosclerosis, cancer, neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, diabetes mellitus, inflammatory diseases, as well as psychological diseases and aging processes (Durackova, 2010).

Role of oxidative stress in cardio-vascular diseases: Cardiovascular disease is the major cause of mortality around the world. It may be broadly classified into coronary heart disease, cerebrovascular disease and peripheral vascular disease, in which the blood supplies to the heart, the brain and the peripheral vasculature, respectively, are compromised (Ross, 1999). Common to each of these classifications is the formation of an atherosclerotic plaque or lesion (an atheroma) which can occlude small blood vessels and disrupt blood flow (Ross, 1993). 

Figure 2: Relationship between radicals and antioxidants

This leads to acute manifestations such as myocardial infarction and stroke in which tissue oxygen and nutrient supply are severely compromised (Rader and Daugherty, 2008). Atherosclerosis is a complex disorder involving numerous cell types which contribute to the initiation and formation of atherosclerotic lesions. Production of oxygen free radicals is the key to the progression of this potentially fatal inflammatory disorder at a number of cellular sites, along with reduced cell antioxidant capacity (Fearon and Faux, 2009). The ROS participate in normal cell signaling as mediators regulating vascular function (Harrison et al., 2003). The loss of myocytes through apoptosis or programmed cell death, has been reported in the infarct regions of myocardium from myocardial infarction patients as well as from patients with end-stage heart failure (Narula et al., 1996). Any acute or chronic cardiac stress conditions, resulting in a relative deficit in the myocardial ‘antioxidant reserve’, are associated with an increase in myocardial ‘oxidative stress’ (Singal et al., 1998). Direct involvement of oxidative stress in apoptosis has been demonstrated in a variety of cell types (Hockenbery et al., 1993). Antioxidant vitamins, such as vitamin C, carotenoids and vitamin E, have been shown to decrease lipid peroxidation and reduce atherogenesis and the risk of coronary heart disease. Vitamin E being a ‘chain breaking’ antioxidant acts to protect polyunsaturated fatty acids from oxidation by interrupting the chain reaction of lipid peroxidation in the membrane (Packer, 1991).

The ROS generated by mitochondria play an important role in signaling pathways which contribute to cardiovascular disease. In few cases the primary stimulus for the activation of these pathways has been identified. The vasoactive agents such as angiotensin II (Kimura et al., 2005), Epidermal Growth Factor (EGF) (Krieg et al., 2004), transforming growth factor-β (TGF-β) (Herrera et al., 2001) and Tumor Necrosis Factor-α (TNF-α) (Chen et al., 2004) are capable of modulating mitochondrial ROS production. The mitochondrial contribution to growth factor signaling response appears to involve the transactivation of growth factor receptors and is associated with the protection against oxidative stress (Chenet al., 2004; Daugherty et al., 1994).

Role of oxidative stress in diabetes: Diabetes Mellitus (DM) is a group of metabolic diseases characterized by abnormal insulin secretion, derangement in carbohydrate and lipid metabolism and is diagnosed by the presence of hyperglycemia. This clinical metabolic syndrome is also characterized by hypertriglyceridemia, reduced HDL (good cholesterol) and abnormal postprandial lipemia finally leading to atherosclerosis. Type 1 diabetes or Insulin-Dependent Diabetes Mellitus (IDDM) is a complex, multifactorial disease involving severe destruction of the insulin-producing pancreatic β-cells. Oxidative stress may play an important role in diabetes, not just because of its role in the development of complications but because persistent hyperglycemia, secondary to insulin resistance, may induce oxidative stress and contribute to β-cell destruction in type 2 diabetes (Mohora et al., 2007). In diabetes, oxidative stress seems caused by both increased production of ROS, sharp reductions in antioxidant defenses and altered cellular redox status (West, 2000; Goldstein et al., 2005). Although the source of this oxidative stress remains unclear, it has been suggested that the chronic hyperglycaemia in diabetes enhances the production of ROS from glucose autoxidation, protein glycation and glycoxidation which leads to tissue damage (Brownlee, 2000). The increase in the level of ROS in diabetes could be due to their increased production and/or decreased destruction by nonenzymic and enzymic catalase (CAT), reduced glutathione (GSH) and superoxide dismutase (SOD) antioxidants (Lipinski, 2001; Slatter et al., 1999; Jakus, 2000).

Role of oxidative stress in carcinogenesis: Cancer is a synonym for malignant neoplasm. It is a genetic disease, a process leading a cell from a healthy to a precancerous state and finally to an early stage of cancer. Cancer development takes place in three major stages: Initiation, promotion and progression. Carcinogenesis involves a cascade of events involving mutation and the subsequent selective clonal expansion of the mutated cell. Cancers evolve by a reiterative process of clonal expansion, genetic diversification and clonal selection within the adaptive landscapes of tissue ecosystems (Greaves and Maley, 2012; Cadet et al., 2010). There exists an intricate inflammatory network. Chronic inflammation is induced by biological, chemical and physical factors and is in turn associated with an increased risk of several human cancers (Bartsch and Nair, 2006). The link between inflammation and cancer has been suggested by epidemiological and experimental data (Grivennikov et al., 2010) and confirmed by anti-inflammatory therapies that show efficacy in cancer prevention and treatment (Gonda et al., 2009). Carcinogens induce carcinogenesis by myriad modes of action, including production of ROS. Initiation of carcinogenesis starts just after an inflammatory stimulus which is either mediated by ROS and may be direct (oxidation, nitration, halogenation of nuclear DNA, RNA and lipids) or may be mediated by the signaling pathways activated by ROS. The H₂O₂, another example of ROS, are short-lived but the most damaging radicals within the body and may be formed either by dismutation from superoxide anion or spontaneously in peroxisomes from molecular oxygen (Ray and Husain, 2002; Bellezza et al., 2010; Hanahan and Weinberg, 2000; Barkett and Gilmore, 1999).

Role of oxidative stress in neurodegenerative diseases (NDD): Brain is one of the most metabolically active organs of the body along with the spinal cord comprising Central Nervous System (CNS) which, even at rest, utilizes an estimated 20% of the total oxygen uptake. Brain is vulnerable to oxidative damage because of its high oxygen utilization, high content of oxidizable polyunsaturated fatty acids and the presence of redox-active metal ions (Markesbery, 1997). The NDD includes Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), cerebellar disorders, Parkinson’s Disease (PD), Huntington’s Disease (HD) and stroke in aged populations. These may be related to the oxidative damage in neurons either primarily or secondarily (Rao and Balachandran, 2002). Oxidative stress plays a ubiquitous role in NDD. However, high levels of oxidative stress can cause necrosis, ATP depletion and prevention of controlled apoptotic death (Beal, 2005). Though CNS has a high requirement for oxygen but unexpectedly, is relatively deficient in the enzymes that metabolize a number of oxygen-based reactants to innocuous species (Nunomura et al., 2006). Being the power house of the cell, mitochondria are directly associated and susceptible to oxidative stress. There has been increasing evidence suggesting that mutations acquired during aging by mtDNA (mitochondrial DNA) contribute to physiological decline occurring with age and age-related neurodegeneration (Lin and Beal, 2006; Shukla et al., 2011). Therefore, oxidative stress is an important factor in neurodegenerative diseases, as the damage of the neurons could be due to either an increase in oxidative process or a decrease in anti-oxidant defenses or both.

Antioxidative therapies: Nutraceutical antioxidants provide strong scientific support to be developed as novel therapies for ameliorating myriad of diseases. Several of these natural antioxidants not only actively scavenge free radicals but they also function as modulators of pro-survival or pro-apoptotic signaling pathways. Henceforth, these natural compounds may elicit a greater potential as effective therapeutic agents than traditional medicines with only one mechanism of action. The advancement of this research may lead to the development of nutritional products (nutraceuticals) and semisynthetic analogs that preserve substantial antioxidant capacity but produce minimal side effects (Vidya and Devasagayam, 2007).

According to a "French Paradox" there exists an interesting co-relation between the healthful and nutritive properties of red wine and cardiovascular health. The French paradox is an incentive for more research in countries with low Coronary Heart Disease (CHD) incidence and probably more protective CHD risk factors (Ferrieres, 2004). After virtually 20 years of research study, there is now escalating evidence that small doses of wine intake would prove to be beneficial for the cardiovascular health, acting through a variety of mechanisms that target all the crucial steps of atherosclerosis, from the early formation of the atherosclerotic plaque to its life-threatening complications viz., ulceration, thrombosis, vessel occlusion and infarction (Lippi et al., 2010). These beneficial effects can be attributed to the synergic properties of several biochemical components of wine (alcohol, resveratrol and especially polyphenolic compounds), particularly the red varieties.

A wide variety of antioxidant compounds derived from natural products (nutraceuticals) have demonstrated neuroprotective activity in either in vitro or in vivo models of neuronal cell death or neurodegeneration, respectively (Kelsey et al., 2010). Antioxidants can thus act as a protective agent in neurodegenerative diseases and can be considered as a promising approach to slowing the progression and limiting the extent of neuronal cell loss in these disorders (Moosmann and Behl, 2002; Kannappan et al., 2011). Some nutraceuticals are well known, like epigallocatechin 3-gallate (EGCG) from green tea and resveratrol from grapes which displays neuroprotective effects (Dugan et al., 2001). Antioxidant nutrients may complement the therapies to reduce oxidative stress during diabetes. Dietary supplementation with antioxidant rich food can help diminish oxidative stress associated with NIDDM. However, chronic diabetic complications can be prevented and cured with certain nutritional antioxidants. Primary among these are vitamin E (α-tocopherol) and lipoic acid (thioctic acid). Vitamin E is a fat-soluble vitamin that effectively scavenges the peroxyl radical in cell membranes, thereby inhibiting lipid peroxidation. Prospective epidemiological studies demonstrate that high serum vitamin E levels are associated with decreased risk of NIDDM (Tapas et al., 2008). In addition to this many other antioxidant nutrients have been reported to be beneficial for subjects with NIDDM. Flavonoids are a class of secondary plant phenolics with significant antioxidant and chelating properties, ubiquitously found in commonly consumed fruits, vegetables and beverages such as red wine and tea. 

Figure 3: Synopsis of free radical formation, oxidative stress and pathogenesis of chronic diseases

They have been demonstrated to protect against oxidative stress in type 1 and 2 diabetes (Salonen et al., 1995). Specifically, the flavonoids inhibit lipid oxidation and delay the depletion of lipid-soluble antioxidants. Serum levels of carotenoids, another group of antioxidant compounds often present in edible plants, were inversely related to fasting serum insulin levels (Lean et al., 1999). The protective effects of flavonoids in biological systems are ascribed to their capacity to transfer electrons free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce alpha-tocopherol radicals and inhibit oxidases (Ford et al., 1999).

Dietary and endogenous antioxidants react with oxidizing free radicals and prevent cellular damage by eliminating them. Involvement of chemotherapeutic agents in cancer treatment may lead to the generation of free radicals causing cellular damage and necrosis of malignant cells. So, a concern arises as to whether exogenous antioxidant compounds taken concurrently during chemotherapy could reduce the beneficial effect of chemotherapy on malignant cells. Oxidative damage to nucleic acids can be declined by supplementation with pure antioxidants or with foods rich in antioxidants (Hirano et al., 2001) which would check the development of malignancies due to mutations. The study of antioxidant use in cancer treatment is a rapidly evolving area. Antioxidants have been extensively studied for their ability to prevent cancer in humans (Singal et al., 1998). Thus, there is a current need for exploring new therapies which would increase the efficacy of cancer treatment. Appropriate use of antioxidants along with chemotherapy may be an effective measure for curing cancer thereby facilitating cancer therapy to the pinnacle of success. Widely characterized anti-tumor flavonoids include- epigallocatechin gallate (from green tea), genistein (from soy and red clover) curcumin (from turmeric), silibinin (from milk thistle) and quercetin (from many yellow vegetables) (Bushman, 1998; Miodini et al., 1999). Nutraceuticals are compounds showing great potential in nutritional industry due to their capacity to combat severe and chronic diseases and preventing the emergence of other diseases. Thus these substances tend to modify body’s physiological processes (Jain and Ramawat, 2013). Synopsis of free radical formation, oxidative stress and pathogenesis of chronic diseases is represented in Fig. 3.

CONCLUSION
Free radicals/ROS/RNS are produced mainly from diverse sources in the biological system causing a cascade of oxidation events leading to disruption of cellular membranes and attacking other major cell organelles. An antioxidant usually works by retarding this process of oxidation caused by these unstable entities. Most of the chronic diseases arise due to improper nutrition or are indirectly related to diet so, a central role is being played by nutrients in the prevention of such ailments.

ACKNOWLEDGMENTS
Sadaf Kalam would like to express her deepest appreciation towards her mentor Prof. Appa Rao Podile, Department of Plant Sciences, University of Hyderabad, for his incessant support and encouragement during the writing of this manuscript. The authors would like to thank Prof. Abul Kalam Department of Linguistics, MANUU, Hyderabad for his critical eval‎uation and suggestions during the drafting of this manuscript. The authors would also like to acknowledge Mr. S.N. Vijaywargia, Chancellor, Peoples’ University, Bhopal, (M.P) for his encouragement and moral support.

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