ANTIOXIDANT AND TOXICOLOGIC PROPERTIES OF METHANOL LEAF EXTRACT OF STEPHANIA DINKLAGEI IN WISTAR ALBINO RAT

ABSTRACT

Stephania dinklagei is used extensively in South East Nigeria for the traditional treatment of malaria and other associated ailments in form of decoction, in which unspecified quantities are usually consumed without due regards to toxicologic and  other adverse effects. In this study,  the  phytochemicals  were  assessed  as  well  as  the  effects  of  the  antioxidant  and toxicologic properties of methanol leaf extract of  stephania dinklagei in Wistar albino rat. The rats were administered with graded doses of the extract twice daily for three weeks and the control administered with distilled water. Four rats each from the control and test groups were sacrificed every seven days and blood samples collected for analysis.  The percentage yield  of stephania  dinklagei  methanol  leaf extract  was  5.5%.  Preliminary  phytochemical screening  showed  that  the methanol  leaf extract  contained  alkaloids,  flavonoids,  tannins, steroids, terpenoids, carotenoids, glycosides, anthocyanins and saponins. Anthraquinone was not  detected.  The  quantitative  phytochemical  analysis  showed  that  the  extract  contains alkaloids (29.70 + 0.15mg/g), flavonoids (25.30 + 0.10mg/g), steroids (69.70 + 0.10mg/g), saponins  (13.57  +  0.21mg/g),  tannins  (64.21+  0.21mg/g)  cardiac  glycosides  (1.45   +

0.09mg/g), terpenoids (44.30 + 0.26mg/g), carotenoids (5.88 + 0.52mg/g) and  anthocyanin

(15.40 + 0.26mg/g). The vitamin content of the leaf extract was found to be vitamin A (44.8

+ 0.42mg/100g), vitamin C (27.85 + 0.07mg/100g) and vitamin E (12.7 + 0.28mg/100g). The acute toxicity test of the leaf extract showed no toxicity up to 5000mg/kg body weight as observed  over  a period  of 48  hrs  for  signs  of acute  toxicity.  The extract  was  found  to moderately scavenge the DPPH and superoxide anion radical in a dose  dependent manner compared  with  their  respective  standards.  The  extract  however,   highly  scavenged  the hydroxyl radical when compared with the standard, α-tocopherol. There were no significant differences  (p >0.05)  in serum  MDA level  in all the groups  in week I but significantly increased (p<0.05) in group 4 (week 2) when compared with that of their control. The serum SOD activity showed a significant decrease (p<0.05) in all the groups of 1st, 2nd and 3rd weeks

of the experiment when compared with that of their respective controls. Serum CAT  also decreased significantly (p<0.05) in group 3 and 4 in week 3 compared with the control but no significant difference (p<0.05) was observed in all the groups in week 1 and 2. Serum ALP activity  increased  significantly  (p<0.05)  throughout  the duration  of  the  experiment  when compared with that of their controls. Serum ALT level increased significantly (p<0.05) only

in group 4 in the 1st, 2nd and 3rd weeks of the experiment. The same trend was observed with

the  AST  level  when  compared  with  those  of  their  controls.  Creatinine  showed  a  non- significant increase (p>0.05) in groups 2 and 3 but significantly decreased (p<0.05) in group

4 (week 1). There were also non-significant difference (p>0.05) in all the groups in week 2 when compared with that of their control but in week 3, there was non-significant increase (p>0.05) in groups 2 and 3 and a non-significant decrease in group 4. Urea level significantly increased (p<0.05) in all the groups throughout the duration of the experiment. Serum Na+ increased significantly (p<0.05) in week 1, 2 and 3 compared with those of their respective controls. Serum Cl-  level showed  non-significant  difference (p>0.05) in week 1 and 2 but however, increased significantly (p<0.05) in week 3 compared with the control. Histological examination of the liver cells of the treated rats revealed widespread hepatocellular vacuolar degeneration  with  hypertrophy  of  kupffer  cells  in  the  periportal  areas  and   moderate infiltration of mononuclear leucocytes into the periportal area as against that of their control. The histopathology result corroborates the results of the serum biochemical parameters. The kidney showed  no significant  changes  in the treated  groups  compared  with that of their

control. These results suggest that Stephania dinklagei leaf extract had a significant in vitro antioxidant  activity.  However,  long term consumption  of the extract at the doses  studied could be hepatotoxic but not nephrotoxic.

CHAPTER ONE

INTRODUCTION

The Southern inhabitants  of Nigeria are known for their high consumption of  vegetables. Some of these vegetables form part of foods consumed on special conditions, including ill health  and  times  of  convalescence.  This  stresses  the  role  of  plants  in  the  life  of  man (Nwangwu  et al., 2009). The use of plant parts in  traditional medical practice has a long drawn  history  and  remains  the  mainstay  of  primary  health  care  in most  of third  world countries (Prescott-Allen, 1982). Medicinal plants are believed to be an important source of some  secondary  metabolites  with  potential  therapeutic  benefits  (Farnsworth,  1989).  In treatment of diseases, the use of herbs has gained grounds world wide, making traditional medicine an inevitable global discuss. This practice calls for research into pharmacological activities of plants secondary metabolites and has improved modern pharmaco therapeutics around the world (Nwaogu et al., 2007). Though, some medicinal plants serve as food, they contain secondary metabolites that influence biological processes and reverse disease states (Ugochukwu and Badaby, 2002).

In normal or pathological cell metabolism, free radicals which have one or more unpaired electrons are produced. Reactive Oxygen Species (ROS) react easily with free radicals such as superoxide anion radical (O2-) and hydroxyl anion (OH-) as well as non-free radical species

(H2O2) and the singlet oxygen (1O2) (Yildrim et al., 2002). Also excessive generation of ROS

induced by various stimuli and which exceed the antioxidant capacity of the organism leads to a variety of pathophysiological processes such as inflammation, diabetes, genotoxicity and cancer  (Kourounakis  et al., 1999).  A great number of medicinal  plants contain chemical compounds  that  exhibit  antioxidant  properties  (Gulcin  et  al.,  2002).  Sources  of  natural antioxidants are primarily plant phenolics that may occur in all parts of plant such as fruits, vegetables, nuts, seeds, leaves and barks (Pratt and Hudson, 1990).

1.1      Stephania dinklagei

Figure 1: Stephania dinklagei leaves

A  large  number  of  alkaloids  have  been  isolated  from  Stephania  dinklagei,  it  contains corydine (sedative drug agent) and Stephanini (analgesic drug agent) (Goren et al., 2003). Its constituent  Liriodenine exhibits antiprotozoal and cytotoxic activities  against the protozoa Leishmania  donovania  and  Plasmodium  falciparum  (Camacho  et  al., 2000).  An infusion made from its young leaves is immediately given to children  before it thickens to relieve them from stomach aches (Goren et al., 2003). Its leaves are taken to treat impotency in men and also act as an aphrodisiac (Burkill, 1997). Given its uses in the traditional setting and the emerging  reports on its pharmacological  actions,  it  is therefore  necessary to evaluate  its antioxidant properties, toxicity and its effect on liver and kidney marker enzymes in Wistar albino rats.

1.2       Phytochemicals

Phytochemicals   are  chemical  compounds  formed  during  the  plants  normal   metabolic processes.  These  chemicals  are often referred  to as secondary metabolites  which include alkaloids,   flavonoids,   coumarins,   glycosides,   gums,  polysaccharides,   phenols,   tannins, terpenes and terpenoids (Harborne, 1973; Okwu, 2004). These can act as agents to prevent undesirable side effects of the main active substances or to assist in the assimilation of main substances (Anonymous, 2007). Phytochemicals are present in a variety of plants utilized as important components of both human and animal diets. These include fruits, seeds, herbs and vegetables (Okeke and Elekwe, 2003). Most of these phytochemical constituents are potent bioactive compounds found in medicinal plant parts which are precursors for the synthesis of useful drugs (Sofowora, 1993).

1.2.1    Alkaloid

Alkaloids  are  group  of  naturally  occurring  low  molecular  weight  nitrogenous  chemical compound  that  contain  mostly  basic  nitrogen  atoms  (Manske,  1965).  They  are  found primarily  in  plants  and  are  especially  common  in  certain  families  of  flowering  plants (Herbert, 1999). Large variety of organisms produce alkaloids, these include bacteria, fungi, plants and animals and are part of the group of natural products called secondary metabolites (Baldwin and Ohnmeiss, 1993). Most alkaloids contain oxygen in their molecular structure, those compounds  are usually colourless  crystals at  ambient  conditions  (Lewis and Elvin- Lewis,  1977).  Oxygen-free  alkaloids,  such  as  nicotine  or  coniine  are  typically  volatile, colourless, oily liquids (Abuo-Donia et al., 1992) some alkaloids are coloured, like berberine (yellow) and sanguinarine (orange) (Akhtar et al., 2003). Most alkaloids are weak bases, but some, such as theobromine  and  theophylline are amphoteric (Ali and Khan, 2008). Many alkaloids dissolve  poorly in water but readily dissolve in organic solvents such as diethyl ether, chloroform or 1, 2-dichloroethane. Caffeine, cocaine, codeine and nicotine are water soluble (Ashihara et al., 2008). Biological precursors of most alkaloids are amino acids such as  ornithine,   lysine,   phenylalanine,   tyrosine,   tryptophan,   histidine,   aspartic   acid   and anthranilic acid (Berkov et al., 2007). Alkaloid biosynthesis are too numerous and cannot be easily classified (Blankenship et al., 2005).

Most of the known functions of alkaloids are related to protection. For example, aporphine alkaloid – liriodenine produced by the tulip tree protects it from parasitic mushrooms. Many alkaloids are used in medicine: Atropine, Codeine, Nicotine and Quinine reserpine are used as anticholinergic, stimulant; antipyretics and antihypertensives respectively (Ashihara et al.,

2008).

1.2.2    Saponin

Saponins are amphipathic glycosides grouped, in terms of phenomenology, by the soap-like foaming they produce when shaken in aqueous solutions  (Francis  et al.,  2002). Saponins consist of a polycyclic aglycones attached to one or more sugar side chains. The aglycone part, which  is also called sapogenin  is either  steroid (C27) or a  triterpene  (C30) (Skene,

2006). The foaming ability of saponins is caused by the combination of a hydrophobic (fat- soluble) sapogenin and a hydrophilic (water soluble) sugar part. Saponins have a bitter taste. Some saponins are toxic and are known as sapotoxin (XU et al., 1996). They are found in most plants, vegetables, beans and herbs (Francis et al., 2002).  Studies have illustrated the beneficial  effects  on blood  cholesterol  levels,  cancer,  bone  health and stimulation  of the immune system (XU et al., 1996). It has also shown that saponins have anti tumor and anti- mutagenic activities and can lower the risk of human cancers by preventing cancer cells from

growing. It was found that saponins may help to prevent colon cancer and as shown in their article “Saponins as anti-carcinogens” published in the Journal of Nutrition (1995).

1.2.3    Steroid

Steroids are organic compounds that contain a characteristic arrangement of four cycloalkane rings that are joined to each other (Kuzuyama and Seto, 2003). Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone and anti-inflammatory drug dexamethasone  (Rosier,  2006). Steroids  are found  in  plants, animals and fungi.  All steroids are made in cells either  from the sterols  lanosterol  (animals and  fungi)  or from cycloartenol (plants). Both lanosterol and cycloartenol are derived from the cyclization of the triterpene  squalene  (Kuzuyama  and  Seto,  2003).  Steroids  have  a chemical  structure  that contains the core of gonane or a  skeleton derived there from. Usually, methyl groups are present at the carbons C-10  and C–13 – an alkyl side-chain at carbon C–17 may also be

present (Dubey, et al., 2003).

H          R

H       H

H          H

Fig. 2: The basic skeleton of a sterHoid, with standard stereo orientation

The three cyclohexane rings form the skeleton of phenanthrene, the last ring of the  gonane has  a  cyclopentane  structure.  Hence,  together  they  are  called   cyclopentaphenanthrene (Hanukoglu,  1992).  Steroid  biosynthesis  is an anabolic  metabolic  pathway that  produces steroids  from  simple  precursors.  A unique  biosynthetic  pathway  is  followed  in  animals compared to many other organisms, making the pathway a common target for antibiotics and anti-infective  drugs  (Hanukoglu,  1992). In addition, steroid  metabolism  in humans is the target  of  cholesterol  lowering  drugs  such  as  statins.  In  humans  and  other  animals,  the biosynthesis  of steroids follows the mevalonate  pathway that uses acetyl-CoA  as  building blocks to form dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) (Dubey et al,  2003).  In subsequent  stages,  DMAPP  and  IPP  are  joined  to form geranyl

pyrophosphate (GPP), which in turn is used to synthesize the steroid lanosterol,  lanosterol can then be converted into other steroids such as cholesterol and ergosterol (Rosier, 2006).

1.2.4    Flavonoid

Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. Flavonoids consist  of 6  major  subgroups:  Chalcone,  flavone,  flavonol,  flavanone,  anthocyanins  and isoflavonoids. Together with carotenes, flavonoids are also responsible for the colouring of fruits, vegetables and herbs (Galeotti et al., 2008).

Flavonoids are synthesized by the phenyl-propanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA  (Cazarolli et al., 2008; Friedman,

2007). This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group  of compounds  called  chalcones,  which  contain  two  phenyl  rings.  Conjugate  ring- closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone (Friedman, 2007). The metabolic pathway continues through a series of enzymatic modifications  to  yield  flavanones  →diydroflavonols→anthocyanins.   Along  this  pathway, many  products  can  be  formed,  including  the  flavonols,  flavan-3-ols,  proanthocyanidins (tannins) and a host of other various polyphenolics (Verueridis et al., 2007). In vitro studies show that  flavanoids also have anti-allergic,  anti-inflammatory,  anti-microbial,  anti-cancer and anti-diarrheal activities (Cushnie and Lamb, 2011). Research has shown that flavonoids are poorly absorbed in the human body (less than 5%), with what is absorbed being quickly metabolized and excreted (Williams et al., 2004).

1.2.5    Tannins

Tannin is an astringent,  bitter plant polyphenolic  compound  that binds to and  precipitates proteins and various other organic compounds including amino acids and alkaloids (Muller- Harvey and McAllan, 1992). Tannin compounds are found mainly in plants where they play a role  in protection  from  predation,  and  perhaps  also  as  pesticides  (Giner-Chavez,  1996). Tannins play an important role in the ripening of fruit and the aging of wine. There are three large classes of secondary metabolites in plants: Nitrogen containing compounds, terpenoids and phenolics (Mole, 1993). Tannins belong to the phenolics class. All phenolic compound (primary and secondary) are, in one way or another formed via the shikimic acid pathway, also known as the phenylpropanoid pathway (Reed, 1995). The same pathway leads to the formation of other phenolics  such as isoflavones,  coumarins,  lignins and aromatic  amino acids (tryptophan,  phenylalnie  and  tyrosine).  Typically,  tannin molecules  require  at least twelve  hydroxyl  groups  and  at  least  five  phenyl  groups  to  function  as  protein  binders.

Tannins  are  important  ingredient  in  the  process  of  tannin  of  leather.  Tannins  produce different atoms with ferric chloride according to the type of tannin (Souza et al., 2006).

1.2.6    Anthocyanin

Anthocyanins are water-soluble vacuolar pigments that belong to a parent class of molecules called flavonoids synthesized via the phenylpropanoid pathway, they are odourless and nearly flavourless  (Stafford,  1994). Anthocyanins  occur in all tissues  of  higher  plants including leaves, stems, roots, flowers and fruits (Wada and Ou, 2002). Anthocyanins have been shown to act as a “sunscreen”, protecting cells from high-light damage by absorbing blue-green and ultraviolet  light,  thereby protecting  the  tissues  from photo  inhibition,  or high-light  stress (Lieberman, 2007). Anthocyanins can be used as pH indicators because their colour changes with pH; they are pink in acidic  solutions, purple in neutral solutions, greenish-yellow  in alkaline solutions and colourless in very alkaline solutions where the pigment is completely reduced (WU et al., 2004). They are found in cell vacuole. The anthocyanins are subdivided into the  sugar-free  anthocyanidin  aglycones  and the anthocyanin  glycosides.  As of 2003, more  than  400  anthocyanins  had  been  reported  (Lieberman,  2007).  While  more  recent literature (early 2006) puts number at more than 550 different anthocyanins. The difference in chemical structure that occurs in response to changes in pH is the reason why anthocyanins are often used as pH indicators, as they change from red in acids to blue in bases (De-Rosso et  al.,  2008).  In  anthocyanin   biosynthetic  pathway,   L-phenylalanine   is  converted   to naringenin  by  phenylalanine  ammonialyase  (PAL),  Cinnamate-4-hydroxylase   (C4H),  4- Coumarate CoA Ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI). And then, the next pathway is catalysed by the formation of complex aglycone and anthocyanin

composition  by  flavone  3  –  hydroxylase  (F3H),  flavonoid  31   –  hydroxylase  (F31H),

dihydroflavonol  4 –  reductase  (DFR).  Anthocyanidin  synthase  (ANS).  UDP –  glucoside: flavonoid glucosyltransferase (UFGT) and methyl transferase (MT). Among these, UFGT is divided into UF3GT and UF5GT, which are responsible for the glucosylation of anthocyanin to  produce  stable  molecules  (WU  et  al.,  2004).  Although  anthocyanins  are  powerful antioxidants in vitro, this antioxidant property is unlikely to be conserved after the plant is consumed (Stafford, 1994).

1.2.7     Cardiac Glycoside

Cardiac  glycosides  are  glycosides  of  mostly  C23  –  steroidal  compounds.  They  have  a characteristic 5 – or 6 – membered lactone ring (Wang et al., 2008). They are called cardiac glycosides because they modify heart action (Brower et al., 1972). Cardenolides inhibit the

Na+ – K + – ATPase pump in mammals. This group of compounds is found in a large number

of families many of which are unrelated. A number of toads and frogs make cardiac active compounds that are steroidal but not glycosidic in nature (Wang et al., 2008). Cardenolides are  derived   from  steroidal  precursors,   probably  cholesterol   via  the   intermediacy   of pregnenolone  or progesterone  intermediates  (Jungreis et al., 1997).  Most members of the family Asclepiadaceae contain cardiac glycosides (Dussourd, 1986). Cardiac glycosides are drugs used in the treatment of congestive  heart failure  and cardiac arrhythmia (Dussourd,

1986). Drugs such as Ouabain and digoxin are cardiac glycosides. Digoxin from the foxglove plant is used clinically, whereas Ouabain is used only experimentally due to  its extremely high potency (Dussourd,  1986).  Normally,  sodium-potassium  pumps in  the membrane  of cells (in this case, cardiac myocytes) pump potassium ions in and sodium ions out. Cardiac glycosides inhibit this pump by stabilizing it in the E2 – P transition state, so that sodium cannot be extruded: intracellular sodium concentration increases. A second membrane ions exchanger, NCX, is responsible for pumping calcium ions out of the cell and sodium ion in (3Na/Ca):  raised  intracellular  sodium  levels  inhibit  this  pump,  so  calcium  ions  are  not extruded and will also begin to build up inside the cell (Jungreis et al., 1997).

1.2.8    Terpenoid

Terpenoid  are large and diverse  class of naturally occurring organic chemicals similar  to terpenes derived  from five-carbon isoprene units assembled  and modified  in  thousands of ways (Yousefbeyk et al., 2014). Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons (Wolinsky, 1973). These lipids can be found in all classes of living things and are the largest group of natural products (Gimelli, 2001). The steroids and sterols in animals are biologically produced from terpenoid precursors. Sometimes terpenoids are added to proteins, e.g. to enhance their attachment to the  cell  membrane,  this  is  known  as  isoprenylation  (Maarse,  1991).  Terpenoids  can  be thought of as modified terpenes,  wherein methyl groups have been moved or removed  or oxygen  atoms  added  (Swan,  1967).  Just  like  terpenes,  the  terpenoids  can  be  classified according to the number of isoprene units used. There are two metabolic pathways of creating terpenoids: Mevalonic acid pathway and MEP/DOXP pathway (Wolinsky, 1973).

1.2.9    Carotenoid

Carotenoids are organic pigments that are found in the chloroplasts of plants and some other photosynthetic organisms like algae, some bacteria and some fungi (Armstrong and  Hearst,

1996). Carotenoids can be produced from fats and other basic organic metabolic  building blocks by all these organisms (Brian, 1991).

Carotenoids are split into two classes, xanthophylls (which contain oxygen) and  carotenes (which are purely hydrocarbons and contain no oxygen) (Unlu et al., 2005; Brian, 1991). The major  role  of carotenoid  in plants and  algae  is that they absorb  light energy  for use in photosynthesis and they protect chlorophyll from photo damage (Kidd, 2011). Carotenoids belong to the category of tetraterpenoids, structurally, carotenoids take the form of a polyene hydrocarbon  chain  which  is  sometimes  terminated  by rings  and  may  or  may  not  have additional oxygen atoms attached (Unlu, et al., 2005). The most common carotenoids include lycopene and the vitamin A precursor, B-carotene. In plants, the xanthophylls lutein is the most abundant carotenoid and its role in preventing age-related eye disease is currently under investigation (Alija, et al., 2004).

1.3      Antioxidants and free radicals

Antioxidants  are molecules  which can safely interact with free radicals and terminate  the chain reaction before vital molecules are damaged (Vertuani et al., 2004). It is therefore an oxidation reaction (Davies,  1995). Oxidation  reactions  can produce free  radicals.  In turn, these  radicals  can start chain reactions  (Stohs  and Bagchi,  1995).  Antioxidants  are often reducing  agents  such  as thiols,  ascorbic  acid  or  polyphenols  (Valko  et al.,  2007).  Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed  when oxygen  interacts  with  certain  molecules  (Knight,  1998;  Stohs and  Bagchi,

1995). Once formed, these highly reactive radicals can start a chain reaction. Their  chief danger  comes  from  the  damage  they  can  do  when  they  react  with  important  cellular components such as DNA, or the cell membrane (Valko et al., 2004). Cells  may function poorly or die if this occurs. To prevent free radicals damage, the body has a defense system of antioxidants (Benzie, 2003; Davies, 1995). A paradox in metabolism is that, while the vast majority of complex life on earth requires oxygen for its existence, oxygen is highly reactive molecule that damages living organisms by producing reactive oxygen species (Valko et al.,

2007). Consequently, organisms contain a complex network of antioxidants metabolism and enzymes  that work together  to prevent  oxidative  damage  to cellular  components  such as DNA, proteins and lipids (Jha et al., 1995). In general, antioxidant systems either prevent these  reactive  species  from being formed  or remove  them before they  can damage  vital components of the cell (Sies, 1997; Davies, 1995). However,  reactive  oxygen species also have useful cellular  functions,  such as redox signaling.  Thus, the function of antioxidant systems is not to remove oxidants entirely but instead to keep them at an optimum level. The reactive oxygen species produced in cells include hydrogen peroxide (H2O2), hypochlorous

acid (HClO) and free radicals such as the hydroxyl radical (.OH) and the superoxide anion

(O2-) (Hirst et al., 2008). The hydroxyl radical is particularly unstable and will react rapidly and  non-specifically  with  most  biological  molecules  (Jha  et  al.,  1995).  This  species  is produced  from hydrogen  peroxide  in metal-catalyzed  redox reactions  such  as the Fenton reaction (Sies, 1997). These oxidants can damage cells by starting chemical chain reactions

such as lipid peroxidation, or by oxidizing DNA or proteins (Vertuani et al., 2004). Damage to DNA can cause mutations and possibly cancer, if not reversed by DNA repair mechanisms (Knight, 1998) while damage to protein causes enzyme inhibition, denaturation and protein

degradation (Davies, 1995).

Ischemic injury Nephrotoxic injury Phagocyte activation HOCl Phagocyte activation

NADPH oxidize Xanthine oxidase Mitochondria

Cl            MPO

Fe2+        Fe3+

•O2+e- →O2•-             H2O2                                       OH•    X

Lipid peroxidation

L-arg.→     NO+.

INOS

GSSG GSH

Figure 3:  Production and elimination oHf OR+OOS (Hirst et al., 2008)

Ishemic

ONOO-                                                        2                2

1.3i.n1juryHydroxyl radical

The hydroxyl radical, .HO, is the neutral form of the hydroxide ion (HO-). Hydroxyl radicals are highly reactive and consequently short-lived; however, they form an important part of radical chemistry (Sies, 1993). Hydroxyl radicals are produced from the decomposition  of hydroperoxides (ROHO) or atmospheric chemistry by the reaction of excited atomic oxygen with water (Reiter and Caneiro, 1997), Hydroxyl radicals are also produced during UV-light dissociation  of  H2O2   and  likely  in  Fenton  chemistry,  where  trace  amounts  of  reduced transition metals catalyze peroxide – mediated oxidations of organic compounds (Sunil et al.,

2013; Reiter and Caneiro, 1997). The hydroxyl radical is often referred to as the “detergent” of the troposphere because it reacts with many pollutants often acting as the first step to their removal (Sies, 1993). It has an important role in eliminating some  greenhouse gases like methane  and  ozone  (Storey  et  al.,  1981).  The  first  reaction  with  many  volatile  organic

compounds (VOCs) is the removal of an hydrogen atom, forming water and an alkyl radical

(R•) (Sies, 1993) •HO + RH → H2O + R•

The alkyl radical will typically react rapidly with oxygen forming a peroxy radical   R• + O2

→ RO2 (Sunil et al., 2013). Hydroxyl radicals can occasionally be produced as a byproduct of immune action. Macrophages and microglia most frequently generate this compound when exposed to very specific pathogens, such as certain bacteria (Sies, 1993; Sunil et al., 2003). The  destructive  action  of  hydroxyl  radicals  has  been  implicated  in  several  neurological autoimmune diseases such has HAND when immune cells become over-activated and toxic to neighbouring  healthy cells (Sies, 1993). The hydroxyl  radical can damage  virtually all types of macromolecules (Sies, 1993; Storey et al., 1981). Unlike superoxide, which can be detoxified  by  superoxide  dismutase,  the  hydroxyl  radical  cannot  be  eliminated  by  an enzymatic reaction (Storey et al, 1981). Mechanisms for scavenging peroxyl radicals for the protection  of cellular  structures  includes  endogenous  antioxidants  such as melatonin  and glutathione and dietary antioxidants such as mannitol and vitamin E. (Reiter and Carneiro, 1997).

1.3.2    Superoxide anion radical

It has the chemical formula O2• -. It is the product of one-electron reduction of molecular (O2), which occurs widely in nature (Holleman and Wibers, 2001). Superoxide is biologically quite toxic and is deployed by the immune system to kill invading microorganisms (Muller et al.,

2007). In phagocytes,  superoxide  is produced  in large quantities by the enzyme  NADPH oxidase for use in oxygen-dependant killing mechanisms of invading pathogens (Miller and Fridovich, 1986). Mutations in the gene coding for the NADPH oxidase  cause an immune deficiency syndrome called chronic granulomatous disease (Muller et al., 2007; Rapoport et al., 1994) characterized  by extreme susceptibility to infection,  especially catalase  positive organisms (Rapoport et al., 1994). Because superoxide is toxic, nearly all organisms living in the presence of oxygen contain isoforms of the superoxide scavenging enzyme, superoxide dismutase (SOD) (Holleman and Wibers, 2011). SOD is an extremely efficient enzyme that catalyzes  the  neutralization  of  superoxide  nearly as fast as the two  can diffuse  together spontaneously in solution (Rapoport et al., 1994).

1.4      Dietary antioxidants

Dietary antioxidants  vitamins C, E and beta carotene are among the most widely  studied vitamins and are group of organic substance present in minute amounts in foods stuffs that are  essentially  for  normal  metabolism  (Bender,  2003;  Kutsky,  1973;  Halliwel  1991). Vitamins also directly scavenge ROS and upregulate the activities of antioxidant enzymes

(Topinka et al., 1989). Vitamin C is considered the most important water-soluble antioxidant in extracellular fluids. It is capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Sies, 1997). Vitamin E is one of the most important antioxidants; it inhibits ROS – induced generation of lipid peroxyl radicals  thereby protecting cells from peroxidation of PUFA in membrane phospho-lipids from oxidative damage of plasma very low density lipoprotein, cellular proteins, DNA and from membrane degeneration (Aruoma,

1998).  Consequently,  a dietary  deficiency  of  vitamin  E reduces  the activities  of  hepatic catalase, GSH peroxidases and glutathione reductase (Fischer – Nielson et al., 1992). Vitamin C has been cited as being capable of regenerating vitamin E (Sies, 1997). Beta carotene and other  carotenoids  are  believed  to  provide  antioxidant  protection  to  lipid  –  rich  tissues. Research suggests beta carotene may work synergistically with vitamin E (Sies, 1997). A diet that is excessively low in fat may negatively affect beta carotene and vitamin E absorption as well as other fat-soluble nutrients. Fruits and vegetables are major sources of vitamin c and carotenoids, while whole grains and high quality properly extracted and protected vegetable oils are major sources of vitamin E (Sies, 1997).

1.5.0    Oxidative stress

As remarkable as our antioxidant defense system is, it may not always be adequate. Oxidative stress reflects an imbalance between the systematic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage (Sies, 1997; Finkel and Holbrook, 2000). Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell,  including proteins, lipids and DNA (Bjelakovic et al.,

2007; Benzie, 2003). Oxidative stress can cause disruptions in normal mechanisms of cellular

signaling. Reactive oxygen species can be beneficial as they are used by the immune system as a way to attack and kill pathogens (Stohs and Bagchi, 1995). Short-term oxidative stress may also be important in prevention of aging by induction of a process named mitohormesis (Bjelakovic  et  al.,  2007).  Oxidative  stress  is  associated  with  increased  production  of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as glutathione (Vertuani et al., 2004). The effects of oxidative stress depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state (Valko et al., 2004). Oxidative damage in DNA can cause cancer. Several antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxide, glutathione reductase, glutathione S-transferase  etc. protect DNA from oxidative stress. It has been proposed that polymorphisms  in  these enzymes are associated  with DNA damage and subsequently the

individual’s risk of cancer susceptibility (Valko et al., 2004; Sies, 1997). The inflammatory response that occurs after strenuous exercise is also associated with oxidative stress (Knight,

1998). During this process, free radicals are produced by neutrophils  to remove  damaged tissue.  As  a  result,  excessive  antioxidant  levels  may  inhibit  recovery  and   adaptation mechanisms (Davies, 1995). Antioxidants supplements may also  prevent  any of the health gains that normally come from exercise, such as increased insulin sensitivity (Knight, 1998;

Benzie, 2003).

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