ESTIMATION OF TOTAL PHENOLICS TOTAL FLAVONOIDS AND IN VITRO ANTIOXIDANT ACTIVITIES OF METHANOL EXTRACT OF VOACANGA AFRICANA ROOT BARK AND ITS FRACTIONS

ABSTRACT

The in vitro antioxidant activities, total phenolic and flavonoid contents of the methanol extract of Voacanga africana root bark and the n-hexane, ethylacetate and n-butanol subfractions of the methanol extract were carried out in this study. The total phenol content was determined by using Folin-Ciocalteau assay while the the totalflavonoid content was determined by using the aluminium chloride colorimetric assay  method. Total antioxidant activities of the  methanol extract and subfractions of the plant were determined in vitro using three models: 1,1 diphenyl-2- picrylhydrazyl (DPPH) radical scavenging assay, phosphomolybdate method and ferric chloride reducing power. The results obtained showed that n-hexane fraction of the V.africana root bark had the highest total phenol – (116.61±95.13 mg GAE/g) followed by that of ethyl acetate fraction. The highest total flavonoid content was exhibited by n-hexane fraction (467.14±44.22 mg QE/g), followed by that of ethyl acetate fraction. Ethylacetate fraction had the highest (31.19±25.29) total antioxidant capacity (TAC). Methanol extract showed the highest DPPH radical scavenging activity (21.00±0.12), while n-hexane fraction showed the highest (43±11.27) ferric reducing antioxidant power (FRAP) followed by ethyl acetate (30.770±11.94). The antioxidant activities of methanol extract and the subfractions therefore, of Voacanga africana

may be responsible for some of the health benefits attributed to the root of this plant.

CHAPTER ONE

INTRODUCTION

Oxidative damage to cellular biomolecules such as lipids, proteins and DNA is thought to play a crucial role in the incidence of several chronic diseases (Dhalla et al., 2000; Vant Veer et al.,

2000).  Flavonoids are  a  group  of polyphenolic  compounds  found  abundantly  in  the  plant kingdom. Interest in the possible health benefit of flavonoids and other polyphenolic compounds has increased in recent years owing to their potent antioxidant and free- radical scavenging activities (Heim et al., 2002)

Most of the physiological impairment, tissues damages, pathological events or diseases affectinghumans have been attributed as a result of by recent scientific studies to the actions of unstable and extremely reactive chemical species called free radicals and/or reactive oxygen species (Tawaha et al., 2007; Sathishkumar et al., 2009; Subhasree et al., 2009; Jang et al.,

2006).

The imbalance between the production of bodily antioxidant defense system and free radical formation results in oxidative stress. Oxidative stress has been implicated in the alteration of genetic  material,  production of  molecular  entities  that  cause  membrane  lipid  peroxidation, decreased membrane fluidity and cell death. The action of the molecular entities called reactive oxygen species (ROS), if not stopped, may lead to accelerated aging, cancer, cardiovascular diseases, neurodegenerative diseases, and inflammation (Neergheen et al., 2006; JayaPrakash et al., 2001; Wong et al., 2006).

Lipid  peroxidation is  one  of the  major  causes  ofdeterioration in  foods  that  results  in  the formation ofpotentially toxic compounds. This has led to the use of synthetic antioxidants, such as butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ) and propylgallate (PG) as food additives to prevent deterioration; however, their use is stringently regulated, due to their potential health risks andtoxicity (Tawaha et al., 2007). Hence, scientists arenow searching for naturally occurring antioxidants inplants as substitutes for synthetic antioxidants (D’Abrosca et al., 2007; Loo et al., 2007; Stanojevićet al., 2009).

Antioxidants protective effect against lipo-peroxidative damage depends on the hydroxyl group in eachmolecule; however, the effectiveness of antioxidantshas been found to be related also to their incorporationrate into cells and their orientation in the bio-membranes (Saija et al., 1994). Natural  antioxidantsendogenous to  food  of  plant  origin  can  scavengereactive  oxygen  and nitrogen species (RONS); evidence suggests that these may be of greatimportance in preventing the onset of oxidativediseases in the human body (Amarowicz et al., 2010).

Plants  are  a  major  source  of  phenolic  compounds,which  are  synthesized  as  secondary metabolites duringnormal development  in  response to  stress conditions,such as  wound  and exposure to UV radiation among other. Plants may contain simple phenolics, phenolicacids, coumarins, flavonoids, stilbenes, hydrolysable and condensed tannins, lignins and lignans. Distribution of phenolics in plants at the tissue, cellularand subcellular levels is not uniform. Insoluble phenolics are found in cell walls, while soluble phenolics are present within the plant cell vacuoles.

Cell wall phenolics may be linked to various cell components such as sugars. Therefore, the nature ofpolyphenol compounds in plants is complex (Maisuthisakul et al., 2008). The beneficial effects of plant phenolics are related to their antioxidant activity, particularly their ability to scavenge free radicals, todonate hydrogen atoms or electrons, or to chelate metalcations. Besides, phenolic compounds contribute largely to the colour and sensory characteristics offruits and vegetables. In addition, phenols participate ingrowth and reproduction processes, and provideprotection against pathogens and predators. At the cellular level, they participate in cell protection against theharmful action of reactive oxygen species (ROS), mainly oxygen free radicals, produced in response toenvironmental stresses such as salinity, drought, heat intensity or mineral nutrient deficiency, becauseof the imbalance between the production andscavenging of ROS in chloroplasts. These cytotoxic activated oxygen species can seriously disrupt normalmetabolism through oxidative damage to lipids, proteins and nucleic acids. Accordingly, plantscontaining  high  concentrations  of  antioxidants  showconsiderable  resistance  to  the oxidative damage causedby the ROS, as shown in the case of salt stressed plants (Meot-Duros and Magne, 2009).

Recovery of antioxidant compounds from plant  materials is typically accomplished through differentextraction techniques taking into account theirchemistry and uneven distribution in the plant matrix.

Solvent extraction is the most frequently used techniquefor isolation of plant antioxidant compounds  (Sultanaet  al.,  2009).  However,  the  extract  yields,  polyphenolic  contents,  and resulting antioxidant activities of theplant materials are strongly dependent on the nature ofextracting solvent and method, due to the presence of different antioxidant compounds of varied chemicalcharacteristics and polarities that  may or  may not  besoluble in a  particular solvent (Sultana et al., 2009 ; Jakopic et al ., 2009). Polar solvents are frequentlyemployed for the recovery of polyphenols from a plantmatrix. The most suitable of these solvents are (hot orcold) aqueous mixtures containing ethanol, methanol,acetone, and ethyl acetate (Sultana et al., 2009).

1.1 Voacanga africana

1.1.1 Profile of Voacanga africana

Voacanga africana is a deciduous, mesophytic plant of the Apocynaceae family found in the tropical rainforest of Nigeria and the Guinea Savanna. A mature Voacanga africana grows about

6m high, not more than 10m with low widely spreading crown distributed mainly in west Africa. It is known locally as kokiyar in Hausa, pete-peteinigbo, kirongasi in Swahili, and ako-dodo in Yoruba. The flowers are white borne in axillary or terminal loosely branched inflorescent. It produces spherical mottled green fruits which occur mainly in pairs with seeds wrapped in yellow pulp. The plant is used to treat leprosy, generalized oedema and as infant tonic (Iwu,1993).

Figure 1: Picture of Voacanga africana Plant

A decoction of the stem bark and root is used to treat mental disorder and the latex is applied to carious teeth. The decoction of the bark is considered analgesic, and is added to embrocating mixtures used as paste during fracture repairs. Root and bark decoctions are also used to treat cardiac spasms. The fruit decoction is used as a disinfectant and the leaf decoction is used for the treatment of asthma in children (Neuwing, 2000). In the South Eastern part of Nigeria, the plant is featured in many healing rituals.Preparation of the extract (Iwu, 1993) is used to induce hallucinations and trance in religious rituals. In Congo, traditional medicine containing extracts ofVoacanga africana are used as anti-amoebial against intestinal amoebiasis, which is one of the current diseases in the tropics accompanied by diarrhea. It has been reported that V. africana has activity against Entamoeba hystolitica in vitro (Crowwell and Otvos, 2004).

V. africana is also used to treat painful hernia. Analysis of root and bark extract of the plant showed the presence of alkaloids including Voacamine, Voacangine and Vobasine (Oliver- Bever, 1986). The leaves and stem decoction of the plant have been implicated in folk medicine for  the  treatment  of  malaria,  diarrhea,  infant  convulsion,  mental  disorder  and  heart  aches (Burkill, 1995).Other compounds found in the plant include Voascritine, Voacamidine, Voacamine, Voaphylline, Vobustine and Voalpolidine which occur in the leaves and tabersonine is a constituent of the seeds (Iwu, 1993). The alkaloid ibogaine is a powerful hallucinogen also

found in Voacangin supporting its use in the treatment of withdrawal symptoms and craving in drug addicts (Correar and Calixto, 1993).

1.2      Plant Phenolic Compounds and Their Classifications

There are about eight thousands kinds of phenolic compounds, which contain aromatic ring in their chemical structure. Scientists in the modern classification tried to split phenols to the simple phenols and polyphenols, and they have suggested that the phenolic acids belong to simple phenols because of their phenol subunit. Flavonoids are considered as a part of the polyphenols, which contain two or three of the phenol subunits. Therefore, those described as polyphenols have more phenol subunit in their chemical structure (King and Young, 1999).

1.2.1   Phenols and Phenolic Acid

Phenolic   acids   contain   carboxylic   acid   in   the   chemical   structure,   and   each   of   the hydroxycinnamic and hydroxybenzoic is a main pillar of phenolic acids as is evident in figure 2. Moreover, scientists have noted that p-coumaric, caffeic, ferulic, and sinapic acids are the main component part(s) of the hydroxycinnamic.

1.2.2   Flavonoids

The molecular weight for flavonoids is low (Figure 3) (Coultate, 1990). Flavane is the main part of flavonoids which contains two benzene rings (A and B) within its chemical structure. As these two rings are connected to each other through pyrane ring (C), so are all flavones, isoflavones, flavonoids, flavonols, flavanones, anthocyanins, and pro anthocyanidins are members of the flavonoids group of compounds according to a new classification.

1.2.3   Anthocyanins

The anthocyanidin is one of the simple structures of the anthocyanins. They consist of an aromatic ring linked to a heterocyclic ring. The heterocyclic ring is connected to the third aromatic ring through a carbon bond (Konczak and Zhang, 2004). Scientists have found that anthocyanins are actually glycoside of anthocyanidins

1.2.4   Tannins

Tannins are natural products which were present in several plant families, and they have large amounts of phenolic rings in their structure. Tannins are classified into two groups: hydrolysable and condensed. Condensed tannins contain flavonoid units with several degrees of condensation, but the hydrolysable tannins are considered a mixture of simple phenols with ester linkages in their structure. There are many factors such as alkaline, mineral acids and enzymes which have

the ability to hydrolyse tannins (Vermerris and Nicholson, 2006).

Figure 2:        Phenolic Acid

Figure 3:        Flavonoid

Figure 4:        Anthocyanins

Figure 5         Tannin

1.3      Extraction of Phenolic Compounds

1.3.1   Solvents

Scientists have studied and analyzed the effect of different types of solvents such as methanol, hexane and ethylacetate in the extraction of antioxidants from of different parts of a plant such as leaves and seeds. The use of solvents, having different polarity have led to the etraxtion of phenolic compounds from plants with a high degree of accuracy (Razali et al., 2012). Anokwuru et al. (2011) found that acetone and N, N dimethylformamide (DMF) are highly effective for the extraction of antioxidants. Relative to ethanol, it has been confirmed that hexane and methanol are more effective for the extraction of phenolics (Anokwuru et al., 2011).However, Razali et al. (2012) found that ethanol extracted higher phenolics from Ivorian plants when compared with acetone, water, and methanol.

1.3.2    Microwave-Assisted Extraction

Microwave-assisted extraction (MAE) has been used as an alternative to conventional techniques for assessing the possibility of reducing both time and volume of extraction solvent used for extracting antioxidants. (Ballard et al., (2010). The advantage of using MAE is the fact that the solvent temperature is raised. The raised temperature enhances the extraction process without loss in solvent volume. (Perez – Sarradilla and Luque de Castro, 2011). So, scientists have endeavoured to promote the usage of this method instead of the other common methods. Li et al., (2012)  reported that  conventional  methods  extracted  less  antioxidant  activity  and  phenolic substances than MAE. They further confirmed that MAE was more effective by comparing the antioxidant activity of extracts obtained by the conventional methods and that of MAE extracts.

1.3.3   Ultrasonic Assisted Extraction (UAE)

Tabaraki and Nateghi (2011) have noted that the current situation of the environment requires the use of green and environmentally friendly technology in order to keep the environment relatively free of toxic substances. Therefore, extraction of phenolic compounds by the ultrasound method has grown significantly during the last few years, in order to reduce the amount of solvent used Corrales et al. (2010) have suggested that UAE has the ability to break down plant tissue and work on the production and release of active compounds into solvents with a high efficiency. Moreover, there is evidence of increased antioxidant activity from 187.13 μmol TE g-1DM to

308μmol  TE  g-1DM  by  using  UAE  as  a  sophisticated  and  effective  method  to  extract antioxidants from different sources.

1.4    Free Radicals, Antioxidants, Diseases and Phytomedicines

Oxygen is an element obligatory to life. Living systems have evolved to survive in the presence of molecular oxygen and for most biological systems. Oxidative properties of oxygen play a Vital role in diverse biological phenomena. Oxygen has double-edged properties, being essential for life, it can also aggravate the change within the cell by oxidative events (Shinde et al., 2006). Free radicals and their adverse effects were discovered in recent times. These are dangerous substances produced in the body along with toxins and wastes which are formed during the normal metabolic processes of the body. The body obtains energy by the oxidation of carbohydrate, fats and proteins through both aerobic and anaerobic processes, which lead to the generation of free radicals.

Overproduction of free radicals can cause tissue injury. Cell membrane are made of unsaturated lipids and these unsaturated lipid molecules of cell membranes are particularly susceptible to free radical effects. Oxidative damage can lead to a breakdown or even hardening of lipids, which are components of all cell walls.Breakdown or hardening is due to lipid peroxidation which leads to death of cells or it becomes impossible  for the cell to properly get its nutrients.

In addition, other biological molecules including RNA, DNA and protein enzymes are also susceptible  to  oxidative  damage.  Environmental agents  also  initiate  free  radical generation leading to various complications in the body. The toxicity of lead, pesticides, cadmium, ionizing radiations, alcohol, cigarette smoke, Uv light and pollution may be due to their free radical initiating capability (Langseth, 1996; Davies, 1991).

Anti-oxidants are substances capable to mopping up free radicals and prevent them from causing cell damage. Free radicals are responsible for causing a wide range of health problems which include cancer, ageing, heart diseases and gastric problems. Anti-oxidants cause protective effect by neutralizing free radicals, which are toxic by-products of natural cell metabolism. The human body naturally produces anti-oxidants but the process is not 100 percent effective in case of overwhelming  production  of  free  radicals  and  that  effectiveness  also  declines  with  age (Goldfarb, 1993).

Increasing the antioxidant intake can prevent diseases and lower the health problems. Research is increasingly showing that anti-oxidant rich foods and herbs contribute to health benefits. Foods may possibly enhance antioxidant levels because foods contain a lot of antioxidant substances.

Fruits and vegetables are loaded with key antioxidants such as Vitamin A,C, E, beta-carotene and important minerals, including selenium and zinc. Fruits, vegetables and medicinal herbs are the richest sources of antioxidant compounds (Sies et al., 1992). Herbs are staging a comeback and herbal “renaissance” is happening all over the world. The herbal products today symbolize safety and most are compatible with human physiology. Natural products, mainly obtained from dietary sources provide a large number of antioxidants. Phyto-constituents are also important sources of antioxidant and capable of terminating free radical chain reactions (Cody et al., 1986; Oluwaseun and Ganiyu, 2008).

1.4 .1  Antioxidants

Antioxidants are substances that delay or inhibits oxidative damage to a target molecule. At a time,  one  antioxidant  molecule can react  with  single  free  radical  and  they are  capable of neutralizing free radicals by donating one of their own electrons, ending the carbon-stealing reaction. Antioxidants prevent cell and tissue damage as they act as scavenger. Cells produce defense against excessive free radicals by their preventive mechanisms, repair mechanisms, physical defenses and antioxidant defenses (Jacob, 2005).

A variety of components act against free radicals to neutralize them from both endogenous and exogenous origin. These include:

    Endogenous enzymatic antioxidants

    Non-enzymatic, metabolic and nutrient antioxidants

    Metal binding proteins like ferritin, lactoferrin, albumin and ceruloplasmin.

    Phytoconstituents and phytonutrients.

The body produces antioxidants (endogenous antioxidants) to neutralize free radicals and protect the body from different diseases caused by the tissue injury. Exogenous antioxidants that are externally supplied to the body through food also plays important role to protect the body. The body has developed several endogenous antioxidant defense system classified into two groups

such as enzymatic and non-enzymatic. The enzymatic defense system includes different endogenous enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx),  glutathione reductase (GR)  and  non-enzymatic defense system included Vitamin  E, Vitamin C and reduced glutathione (GSH) (Jacob, 2005; Harris, 1992).

SOD is an important endogenous antioxidant enzyme which acts as the first line of defense against reactive oxygen species (ROS), particularly superoxide radicals. GPx present in the cytoplasm of cells removes H2O2 by coupling its reduction to H2O with oxidation of GSH. GR is a flavoprotein enzyme, which regenerates GSH from oxidized glutathione in the presence of NADPH. GSH is a tripeptide and a powerful antioxidant present within the cytosol of cells and is the major intracellular non-proteinthiol compound (NPSH). Thiol (SH) groups present in GSH can react with H2O2 and the OH·radical and hence prevent tissue damage. GSH is also capable of scavenging ROS directly or enzymatically via GPx. Vitamins C and E which are non-enzymatic endogenous antioxidant also exist within normal cells and react with free radicals to become radicals themselves which are less reactive than the parent radicals. They break radical chain

reactions by trapping peroxyl and other reactive radicals (Ali et al., 1996; Willcox et al., 2004).

Non-enzymatic antioxidants can also be divided into metabolic antioxidants and nutrient antioxidants. Metabolic antioxidants are the endogenous antioxidants, which are produced during metabolism  in  the  body.  Examples  are  lipoid  acid,  glutathione,L-arginine, coenzyme  Q10, melatonin, uric acid, bilirubin, metal-chelating proteins, transferrin, e.t.c (Willcox et al., 2004; Droge, 2002). Nutrient antioxidants belong to exogenous antioxidants, which cannot be produced in the body but provided through diet or supplements. Examples are trace metals (selenium, magnesium, zinc). Flavonoids, omega-3 and omega-6 fatty acids etc (Pham-Huy et al., 2008). Vitamin E and C are the non-enzymatic antioxidants that exists within normal cells as well. They can be supplied through diet or they can be synthesized endogenously depending on the organc action (Tiwari, 2001).

Antioxidants may exert their actions by several mechanisms. They can suppress the production of reactive oxygen species by reducing hydroperioxides and H2O2. They can also sequester metal ions and, terminate chain reaction by scavenging active free radicals. They and also causerepair and/or clearance of damaged cells. Biosynthesis of other antioxidants or defense enzymes are

also induced by some antioxidants (Tiwari, 2001; Tiwari, 2004). Therefore antioxidant synthesized in the body or supplied from outside like phytoconstituents play important role to protect the body from free radical induced injury.

1.4.2   Free Radicals, Reactive Oxygen and Nitrogen Species

A free radical may be defined as a molecule or molecular fragment containing one or more unpaired electrons in its outermost atomic or molecular orbital and are capable of independent existence (Halliwell and Gutteridge, 1999). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are described as free radicals and other non-reactive derivatives. The reactivity of radicals is generally stronger than non-radical species though radicals are less stable (Pham-Huy et al., 2008). Free radicals are formed from molecules by the hemolytic cleavage of a chemical bond and via redox reactions.Once formed, these highly reactive radicals can start a chain reaction (Bahorun et al., 2006; Valko et al., 2006).

ROS and RNS includes radicals such as superoxide (O2•−), hydroxyl (OH•), hydroperoxyl (HO2•), alkoxyl (RO•), peroxyl (ROO•), nitric oxide (NO•), nitrogen dioxide (NO2•) and lipid peroxyl (LOO•); and non radicals like hydrogen peroxide (H2O2), hypochlorous acid (HOCl), ozone (O3), singlet oxygen (1Δg), peroxynitrate (ONOO−), nitrous acid (HNO2), dinitrogen trioxide (N2O3), lipid peroxide (LOOH) (Pham-Huy et al., 2008). Non radicals are also termed as oxidants and

capable to lead free radical reactions in living organisms easily. Radicals are derived from oxygen characterize as the most important class of radical species generated in living systems (Valko et al., 2006; Miller et al., 1990). At high concentrations, ROS can be important mediators of damage to cell structures, nucleic acids, lipids and proteins (Valko et al., 2007).  O2• − radical is responsible for lipid peroxidation and also have the capability to decrease the activity of other antioxidant defense system enzyme such as catalase (CAT) and glutathione peroxide (GPx), it causes damage to the ribonucleotide which is required for DNA synthesis. The protonated form of O2•− is HO2•, which is more reactive and able to cross the membrane and causes damage to

tissue. OH• radical is most reactive chemical species. It is a potent cytotoxic agent and able to

attack and damage almost every molecule found in living tissue. H2O2  is not a radical but it produces toxicity to cell by causing DNA damage, membrane disruption and release of calcium ions within cell, resulting in calcium dependent proteolytic enzyme to be activated. HOCl is produced by the enzyme myeloperoxidase in activated neutrophils and initiates the deactivation

of antiproteases and activation of latent proteases leading to tissue damage. It has ability to damage biomolecules directly and also decomposes to liberate toxic chlorine. Metal induced generation of ROS attack DNA and other cellular components involving polyunsaturated fatty acid residues of phospholipids, which are extremely sensitive to oxidation (Siems et al., 1995). Peroxyl radicals causes damage after rearranged via a cyclisation reaction to endoperoxides. Studies show that free radicals produce oxidation of the side chains of all amino acid residues of proteins, particularly cysteine and methionine (Valko et al.,2004; Stadman, 2004).

1.4.3   Free Radical Reactions

Free radicals are generally involved in chain reactions, which lead to the regeneration of other radicals that  can begin a  new cycle  of reactions.  Free  radical reactions take three  distinct identifiable steps (Manavalan and Ramasamy, 2001).

    Initiation step: formation of radicals.

      Propagation step: in this step required free radical is regenerated repeatedly as a result of chain reaction, which would take the reaction to completion.

    Termination step: destruction of the generation of free radicals and their sources.

1.4.4   Generation and Sources of Free Radicals

Free radicals can be formed from both endogenous and exogenous substances. They are continuously forming in cell and environment. Different sources of free radicals are as follows (Nagendrappa, 2005; Cadenas, 1989):

    UV radiations, X-rays, gamma rays and microwave radiation.

    Metal-catalyzed reactions.

    Oxygen free radicals in the atmosphere considered as pollutants.

    Inflammation initiates neutrophils and macrophages to produce ROS and RNS.

    Neutrophils stimualated by exposure to microbes.

      In mitochondria-catalyzed electron transport reactions, oxygen free radicals produced as by product.

      ROS  formed  from several  sources  like  mitochondrial cytochrome  oxidase,  xanthine oxidases, neutrophils and by lipid peroxidation.

      ROS  generated  by  the  metabolism  of  arachidonic  acid,  platelets,  macrophages and smooth muscle cells.

      Interaction with chemicals, automobile exhausts fumes, smoking of cigarettes, cigars, beedie.

    Burning of organic matter during cooking, forest fires, volcanic activities.

      Industrial effluents, excess chemicals, alcoholic intake, certain drugs, asbestos, certain pesticides and herbicides, some metal ions, fungal toxins and xenobiotics.

1.4.5   Oxidative Stress and Human Health

Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and metabolism. They are continuously produced by the body via enzymatic and non-enzymatic  reactions  like  respiratory  chain  reaction,  the  phagocytosis,  prostaglandin synthesis, cytochrome p450 system and oxidative phosphorylation (i.e. aerobic respiration) in the mitochondria (Tiwari, 2004).

ROS and RNS are the products of normal cellular metabolism, having both deleterious and beneficial effect in the body (Valko et al., 2004). At low or moderate concentration some of the free radicals plays beneficial physiological role in vivo this include defense against infectious agents by phagocytosis, energy production, cell growth, function in different cellular signaling systems and the induction of a mitogenic response at low concentrations (Poli et al., 2004). Free radicals occur continuously in all cells as part of normal function. Oxygen free radicals are detrimental to the integrity of biological tissue and mediate their injury.

The mechanism of damage involves lipid peroxidation, which destroys cell structures, lipids, proteins  and  nucleic  acids.  They  cause  damage  to  cell  membranes  with  the  release  of intracellular components, leading to further tissue damage (Poli et al., 2004). Antioxidant enzymes and non-enzymatic defense system minimizes the harmful effect of ROS by various antioxidant mechanism. Oxidative stress is a harmful condition that occurs when there is an excess of ROS and/or a decrease in antioxidant levels,  this  may caused tissue damage  by

physical, chemical, psychological factors that lead to tissue injury in human and causes different diseases (Tian et al., 2007). Living creatures have evolved a highly complicated defense system and  body  act  against  free  radical-induced  oxidative  stress  involve  by  different  defense mechanism like preventative mechanisms, repair mechanisms, physical defenses and antioxidant defenses (Valko et al., 2007).

Oxygen derived free radical reactions have been implicated in the pathogenesis of many human diseases  including  neurodegenerative disorder  like  alzheimer’s  disease,  parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, memory loss and depression, cardiovascular disease like atherosclerosis, ischemic heart disease, cardiac hypertrophy, hypertension, shock and trauma,  pulmonary disorders  like  inflammatory  lung  diseases  such  as  asthma  and  chronic obstructive pulmonary disease, diseases associated with premature infants, including bronchopulmonary, dysplasia, periventricular leukomalacia, intraventricular hemorrhage, retinopathy of prematurity and necrotizing enterocolitis, autoimmune disease like rheumatoid arthritis, renal disorders like glomerulonephritis and tubulointerstitial nephritis, chronic renal failure,  proteinuria,  uremia,  gastrointestinal  diseases  like  peptic  ulcer,  inflammatory  bowel disease and colitis, tumors and cancer like lung cancer, leukemia, breast, ovary, rectum cancers etc.  ratina,  maculopathy.  Others  include  ageing  process,  diabetes,  skin  lesions, immunodepression, liver disease, pancreatitis, AIDS, infertility (Pham-Huy et al., 2008; Valko et al., 2007, Agarwal and Prabakaran, 2005).

1.4.6   Phytomedicine as Antioxidants

Human body system is enriched with natural antioxidants and can prevent the onset as well as treat diseases caused and/or fostered due to free-radical mediated oxidative stress. Human also takes antioxidants through diet. In foods, antioxidants found in small quantities are able to prevent or greatly retard the oxidation of easily oxidizable materials (Tiwari, 2001).

Recent researches have shown that the antioxidants of plant origin with free-radical scavenging properties could  have great  importance as therapeutic agents in several diseases caused by oxidative stress (Ramchoun et al., 2009). Plant extracts and phytoconstituents have been found effective as radical scavengers and inhibitors of lipid peroxidation (Dash et al., 2007). Many

synthetic antioxidant compounds have shown toxic and/or mutagenic effects, which have encouraged many investigators to search for natural antioxidant (Nagulendran et al., 2007). Herbal medicine is still the  mainstay of about 75-80% of the world population, mainly in developing countries, for primary health care because of better cultural acceptability,  better compatibility with the human body and lesser side effects. The chemical constituents present in the herbal medicine or plant are a part of the physiological functions of living flora and hence they are believed to have better compatibility with human body.

Natural products from plants are a rich resource used for centuries to cure various ailments. The use of bioactive plant-derived compounds is on the rise.The main reason is the fear that synthetic drugs have their side effects which can be even more dangerous than the diseases they claim to cure. In contrast, plant derived medicines are based upon the premise that they contain natural substances that can promote health and alleviate illness and proved to be safe, better patient tolerance, relatively less expensive and globally competitive. Hence, an appreciation of the healing power of plants and a return to natural remedies as an absolute requirement of better health in the present time (Ramchoun et al., 2009).

Even synthetic drugs used to treat various disorders are capable of producing free radical which leads to oxidative stress and caused tissue damage. For example, non steroidal anti-inflammatory drugs (NSAIDs) are used widely in the treatment of pain, fever, inflammation, rheumatic and cardiovascular disease but chronic administration of those drugs leads the generation of free radicals which may results gastric erosions, gastric or duodenal ulceration and severe complications such as gastrointestinal hemorrhage and perforation (Ramchoun et al., 2009). The use of phytoconstituents as drug therapy to scavenge free radicals and to treat disorders leads due to oxidative stress has proved to be clinically effective and relatively less toxic than the existing drugs. Therefore it is very appreciable and worthwhile of time to uses drugs from plant sources or phytoconstituents to prevent and/or treat oxidative stress (Nagulendran et al., 2007).

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