water extractable phytochemicals from...

Available at: http://www.ictp.it/˜pub_off IC/2006/010

United Nations Educational Scientific and Cultural Organization and

International Atomic Energy Agency

THE ABDUS SALAM INTERNATIONAL CENTRE FOR THEORETICAL PHYSICS

WATER EXTRACTABLE PHYTOCHEMICALS FROM CAPSICUM PUBESCENS (TREE PEPPER) INHIBIT LIPID PEROXIDATION INDUCED BY

DIFFERENT PRO-OXIDANT AGENTS IN BRAIN

G. Oboh* Biochemistry Department, Federal University of Technology,

P.M.B. 704 Akure, Ondo State, Nigeria, Departamento de Quimica, Universidade Federal de Santa Maria (UFSM),

Campus Universitário – Camobi, 97105-900 Santa Maria RS, Brasil and

The Abdus Salam International Centre for Theoretical Physics, Trieste, Italy

and

J.B.T. Rocha

Departamento de Quimica, Universidade Federal de Santa Maria (UFSM), Campus Universitário – Camobi, 97105-900 Santa Maria RS, Brasil.

MIRAMARE – TRIESTE

March 2006

___________________________ * Junior Associate of ICTP. Corresponding author: [email protected]

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ABSTRACT

Reactive oxygen species (ROS) is the cause of neurodegenerative disorders such as Lou

Gehrig's disease, Parkinson's disease and Huntington's disease; one practical way to

prevent and manage neurodegenerative diseases is through the eating of food rich in

antioxidants (dietary means). In this study, the antioxidant and neuroprotective properties

of aqueous extract of ripe and unripe Capsicum pubescens (popularly known as tree

pepper) on different pro-oxidant induced lipid peroxidation in Rat’s brain (in vitro) is

been investigated. Aqueous extract of freshly harvested pepper was prepared, and the

total phenol content, vitamin C, ferric reducing antioxidant property (FRAP) and Fe (II)

chelating ability was determined. In addition, the ability of the extracts to protect the

Rat’s brain against some pro-oxidant (FeSO4, Sodium nitroprusside and Quinolinic acid)

– induced oxidative stress was also determined. The results of the study revealed that

ripe Capsicum pubescens had a significantly higher (P<0.05) total phenol [ripe

(113.7mg/100g), unripe (70.5mg/100g)] content and ferric reducing antioxidant property

than the unripe pepper. However, there was no significant difference in the vitamin C

[ripe (231.5µg/g), unripe (224.4µg/g)] content and Fe (II) chelating ability. Furthermore,

the pepper extracts caused a significant decrease (P<0.05) in 25µM Fe(II), 7µM Sodium

Nitroprusside and 1mM Quinolinic acid induced lipid peroxidation in the Rat’s brain in a

dose-dependent manner. However, the ripe pepper inhibited MDA (Malondialdehyhide)

production in the Rat’s brain than the unripe pepper. Conversely, both extract did not

significantly inhibit Fe (II)/ H2O2 induced decomposition of deoxyribose. Therefore, ripe

and unripe Capsicum pubescens would inhibit lipid peroxidation in vitro. However, the

ripe potent was a more potent inhibitor of lipid peroxidation, which is probably due to its

higher vitamin C and phenol content, reducing power and Fe (II) chelating ability .

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INTRODUCTION

Pepper is an important agricultural crop, not only because of its economic importance,

but also for the nutritional value of its fruits, mainly because they are an excellent source

of natural colours and antioxidant compounds (Howard et al. 2000; Lee et al. 1995). The

intake of these compounds in food is an important health-protecting factor. They have

been recognized, as being beneficial for prevention of widespread human diseases. A

wide spectrum of antioxidant vitamins and phenolic compounds are present in pepper

fruits. Phenol compounds retard or inhibit lipid autoxidation by acting as radical

scavengers (Namiki, 1990); the antiradical activity of phenolics is principally based on

the redox properties of their hydroxy groups and the structural relationships between

different parts of their chemical structure (Rice-Evans et al. 1996; Rice-Evans et al.

1997). Consequently, they are essential antioxidants that protect against propagation of

the oxidative chain. It is also known that pepper fruits contain vitamin C. Vitamin C can

chelates heavy metal ions (Namiki, 1990), reacts with singlet oxygen and other free

radicals, and suppresses peroxidation (Bielski et al. 1975), reducing the risk of

arteriosclerosis, cardiovascular diseases, and some forms of cancer (Harris, 1996).

Reactive oxygen species (ROS), which include free radicals such as superoxide

anion radicals, hydroxyl radicals and non free-radical species such as H2O2 and singled

oxygen, are various forms of activated oxygen (Gulcin et al. 2002; Halliwell and

Gutteridge, 1999; Yıldırım et al. 2001). These molecules are exacerbating factors in

cellular injury and aging process (Lai et al. 2001). In living organisms, various ROS can

be formed in different ways. Normal aerobic respiration stimulates polymorphonuclear

leukocytes and macrophages, and peroxisomes appear to be the main endogenous sources

of most of the oxidants produced by cells.

Exogenous sources of ROS include tobacco smoke, certain pollutants, organic

solvents, and pesticides (Davies, 1994; Halliwell and Gutteridge, 1989; Robinson et al.

1997). ROS can cause lipid peroxidation in foods, which leads to the deterioration of the

food (Miller et al. 1995; Sasaki et al. 1996). In addition, reactive oxygen species induce

some oxidative damage to biomolecules like lipids, nucleic acids, proteins and

carbohydrates. Their damage causes ageing, cancer, and many other diseases (Aruoma,

1994). As a result, ROS have been implicated in more than 100 diseases, including

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malaria, acquired immunodeficiency syndrome, heart disease, stroke, arteriosclerosis,

diabetes, and cancer (Alho and Leinonen, 1999; Duh, 1998; Hertog et al. 1993; Tanizawa

et al. 1992; Yıldırım et al. 2001).

Iron an essential metal for normal cellular physiology can result in cell injury

when in excess. This is because it plays a catalytic role in the initiation of free radical

reactions. The mechanism by which iron can cause this deleterious effect is that Fe (II)

can react with hydrogen peroxide (H2O2) to produce the hydroxyl radical (OH•) via the

Fenton reaction, whereas superoxide can react with iron(III) to regenerates iron(II) that

can participate in the Fenton reaction (Harris et al. 1992; Fraga and Oteiza, 2002).

Sodium nitroprusside is an anti-hypertensive drug, it acts by relaxation of vascular

smooth muscle; consequently it dilates peripheral arteries and veins. However, Sodium

nitroprusside (SNP) has been reported to cause cytotoxicity through the release of

cyanide and/or nitric oxide (NO) (Arnold et al 1984; Bates et al. 1990). NO is a free

radical with short half-life (< 30 s). Although NO acts independently, it also may cause

neuronal damage in cooperation with other reactive oxygen species (ROS) (Halliwell and

Gutteridge, 1999).

Quinolinic acid (QA) is a neuroactive metabolite of the tryptophan-kinurenine

pathway, which can be produced by macrophages and microglia (Cammer, 2001). It is

present in both the human and rat brain (Wolfensberger et al. 1983) and it has been

implicated in the pathogenesis of a variety of human neurological diseases (Belle et al.

2004). Quinolinic acid is recognized pharmacologically as an endogenous glutamate

agonist with a relative selectivity for the NMDA receptor in the brain (Vega-Naredo et al.

2005). Since it is not readily metabolized in the synaptic cleft, it stimulates the NMDA

receptor for prolonged periods. This sustained stimulation results in opening of calcium

channels causing Ca2+ influx followed by Ca2+-dependent enhancement of free radical

production leading to molecular damage and often to cell death (Cabrera et al. 2000).

The brain and nervous system are thought to be particularly vulnerable to

oxidative stress due to limited antioxidant capacity (Vega-Naredo et al. 2005). The brain

is responsible for about two percent of a person's mass but consumes 20 percent of their

metabolic oxygen. The vast majority of this energy is used by the neurons (Shulman et al.

2004). Of particular importance, neurons cannot synthesize glutathione, a fundamental

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component of aerobic cell antioxidant machinery, but instead rely on surrounding

astrocyte cells to provide useable glutathione precursors (Shulman et al. 2004). Because

the brain has limited access to the bulk of antioxidants produced by the body, neurons are

the first cells to be affected by a shortage of antioxidants, and are most susceptible to

oxidative stress (Shulman et al. 2004).

Capsicum pubescens commonly known as tree pepper, its distinctive thick-fleshed

pungent fruits are used as a vegetable condiment or made into a sauce (Brown, 1995;

Facciola, 1990). As a hot pungent flavour, it is mainly used as flavouring in cooked foods

(Brown, 1995). In Peru the seeds are removed, the fruit stuffed with a savoury filling and

then baked (Facciola, 1990). The fruit can be dried and ground into a powder for use as a

pepper-like condiment (Brown, 1995). The hot and pungent fruit is antihaemorrhoidal

when taken in small amounts, antirheumatic, antiseptic, diaphoretic, digestive, irritant,

rubefacient, sialagogue and tonic (Chiej, 1984; Brown, 1995). It is taken internally in the

treatment of the cold stage of fevers, debility in convalescence or old age, varicose veins,

asthma and digestive problems (Brown, 1995). Externally it is used in the treatment of

sprains, unbroken chilblains, neuralgia, pleurisy etc. (Brown, 1995). However, there are

limited information with regard to the ability of the pepper to protect brain against

oxidative stress, this study therefore sought to determine and compare the antioxidant

activity and neuro-protective ability of ripe and unripe Capsicum pubescens against

different pro-oxidant induced oxidative stress in Rat’s brain- In vitro.

MATERIALS AND METHODS

Materials

Fresh samples of ripe and unripe Capsicum pubescens were collected from a vegetable

garden in Camobi, Santa Maria RS, Brazil. The authentication of the pepper was carried

out in Departamento de Biologia, Universidade Federal de Santa Maria, Santa Maria RS,

Brazil. All the chemicals used were analytical grade, while the water was glass distilled.

The handling and the use of the animal were in accordance with NIH Guide for the care

and use of laboratory animals. In the experiments Wister strain albino rats weighing 200

– 230g were collected from the breeding colony of Departamento de Biologia,

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Universidade Federal de Santa Maria, Santa Maria RS, Brazil. They were maintained at

25oC, on a 12h light/12h dark cycle, with free access to food and water.

Aqueous extract preparation

The inedible portion of the pepper were removed from the edible portion, this edible

portion was subsequently washed in distilled water. The aqueous extract of the fresh

pepper was subsequently prepared by homogenizing 1g of the pepper in 20ml-distilled

water, and the homogenates were centrifuge at 2000rpm for 10 minutes. The supernatant

was used for the assay.

Total Phenol Determination

The total phenol content was determined by adding 0.5ml of the aqueous extract of the

pepper to 2.5ml, 10% Folin-Cioalteu’s reagent (v/v) and 2.0ml of 7.5% Sodium carbonate

was subsequently added. The reaction mixture was incubated at 45oC for 40min, and the

absorbance was measured at 765nm in the spectrophotometer, Gallic acid was used as

standard phenol (Singleton et al. 1999).

Vitamin C Determination

The vitamin C was determined using the method of Benderitter et al. (1998), briefly 75µl

of DNPH (2g of dinitrophenyl hydrazine, 230mg thiourea and 270mg in 100ml 5M

H2SO4) was added to 500µl reaction mixture ( 300µl of the pepper extracts with 100µl

13.3% trichloroacetic acid and water respectively). The reaction mixture was

subsequently incubated for 3 hr at 37oC, then 0.5 ml H2SO4 65% (v/v) was added to the

medium, and the absorbance was measured at 520nm, and the Vitamin C content of the

sample was subsequently calculated, using vitamin C standard curve.

Fe2+ Chelation assay

The ability of the aqueous extract to chelate Fe2+ was determined using a modified

method of Minotti and Aust (1987) with a slight modification by Puntel et al. (2005).

Briefly 150µl of freshly prepared 500µM FeSO4 was added to a reaction mixture

containing 168µl of 0.1M Tris-HCl (pH 7.4), 218µl saline and the aqueous extract of the

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pepper (0 - 25µl). The reaction mixture was incubated for 5min, before the addition of

13µl of 0.25% 1, 10-Phenanthroline (w/v). The absorbance was subsequently measured at

510nm in the spectrophotometer

Degradation of Deoxyribose (Fenton´s Reaction)

The ability of the aqueous extract of the pepper to prevent Fe2+/ H2O2 induced

decomposition of deoxyribose was carried out using the method of Halliwell and

Gutteridge (1981). Briefly, freshly prepared aqueous extract (0 – 100µl) was added to a

reaction mixture containing 120µl 20mM deoxyribose, 400µl 0.1M phosphate buffer,

40µl 20mM hydrogen peroxide and 40µl 500µM FeSO4, and the volume for made to

800µl with distilled water. The reaction mixture was incubated at 37oC for 30min, and

the reaction was stop by the addition of 0.5ml of 2.8% TCA (Trichloroacetic acid), this

was followed by the addition of 0.4ml of 0.6% TBA solution. The tubes were

subsequently incubated in boiling water for 20min. The absorbance was measured at

measured at 532nm in spectrophotometer.

Reducing Property

The reducing property of the pepper extracts were determined by assessing the ability of

the extract to reduce FeCl3 solution as described by Pulido et al. (2000), briefly 2.5ml

aliquot was mixed with 2.5ml, 200mM Sodium phosphate buffer (pH 6.6) and 2.5ml of

1% Potassium ferricyanide. The mixture was incubated at 50oC for 20minutes, thereafter

2.5ml, 10% Trichloroacetic acid was added, and subsequently centrifuged at 650rpm for

10 minutes, 5ml of the supernatant was mixed with equal volume of water and 1ml of

0.1% ferric chloride, the absorbance was later measured at 700nm, a higher absorbance

indicates a higher reducing power.

Preparation of Brain Homogenates

The rats were decapitated under mild diethyl ether anaesthesia and the cerebral tissue

(whole brain) was rapidly dissected and placed on ice and weighed. This tissue were

subsequently homogenized in cold saline (1/10 w/v) with about 10-up-and –down strokes

at approximately 1200 rev/min in a Teflon –glass homogenizer. The homogenate was

centrifuge for 10min at 3000xg to yield a pellet that was discarded and a low-speed

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supernatant (S1) containing mainly water, proteins and lipids (cholesterol, galactolipid,

individual phospholipids, gangliosides), DNA and RNA was kept for lipid peroxidation

assay (Belle et al. 2004).

Lipid peroxidation and thiobarbibutric acid reactions

The lipid peroxidation assay was carried out using the modified method of Ohkawa et al.

(1979), briefly 100µl S1 fraction was mixed with a reaction mixture containing 30µl of

0.1M pH 7.4 Tris-HCl buffer, pepper extract (0 - 100µl) and 30µl of the pro-oxidant

solution (250µM freshly prepared FeSO4, 70µM Sodium nitroprusside and 1mM

Quinolinic acid). The volume was made up to 300µl by water before incubation at 37oC

for 1h. The colour reaction was developed by adding 300µl 8.1% SDS (Sodium doudecyl

sulphate) to the reaction mixture containing S1, this was subsequently followed by the

addition of 600µl of acetic acid/HCl (pH 3.4) mixture and 600µl 0.8% TBA

(Thiobarbituric acid). This mixture was incubated at 100oC for 1h. TBARS

(Thiobarbituric acid reactive species) produced were measured at 532nm and the

absorbance was compared with that of standard curve using MDA (Malondialdehyde).

Analysis of data

The result of the replicates were pooled and expressed as mean±standard error (S.E.)

(Zar, 1984); student t-test was carried out. Significance was accepted at P≤ 0.05. The

EC50 (extract concentration that will cause 50% inhibition of lipid peroxidation in the

Rat’s brain) of the pepper extracts were determined using the method of Dixon & Massey

(1969).

RESULTS AND DISCUSSION

Oxidative stress has been extensively studied in neurologic disease including Alzheimer’s

disease, Parkinson’s disease, multiple sclerosis, AIDS dementia and so on. This is not

surprising since the brain (neural cells) is especially susceptible to oxidative stress and

subsequent damage to cells (including cell death). In a disease such as Alzheimer’s,

oxidative stress / oxidative damage are felt to play a key role in the loss of neurons and

the progression to dementia. The most likely and practical way to fight against

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degenerative diseases is to improve the body’s antioxidant status, which could be

achieved by a greater consumption of vegetables (Oboh, 2004; Oboh, 2005a). Ability of

Capsicum pubescens to prevent different pro-oxidant induced oxidative stress in Rat’s

brain (In vitro) are highlighted below.

The results of the total phenol and vitamin C content of the pepper are presented

in table 1. The results revealed that there was no significant difference (P>0.05) in the

vitamin C content of the ripe and unripe Capsicum pubescens [ripe (231.5 µg/g), unripe

(224.4µg/g)]. However, the vitamin C content was lower than that of Capsicum annum,

Tepin [ripe (332.5 µg/g) & unripe (250.2 µg/g)], but higher than that of Capsicum

chinese, Habanero [ripe (152.5 µg/g) & unripe (103.6µg/g)] (Oboh et al. 2005).

Moreover, these values were generally lower than the vitamin C content of Capsicum

annuum L cv. Vergasa (Marin et al. 2004) and what Chu et al. (2002) reported for red

pepper. Nevertheless, the vitamin C content of the pepper was above that of lettuce,

cucumber, celery, cabbage, onion, carrot and some tropical green vegetables (Chu et al.

2002; Oboh, 2005b). Furthermore, the vitamin C content of this pepper is above that of

pineapple, banana, apple, but lower than that of orange, lemon and strawberry (Sun et al.

2002).

However, the ripe pepper had higher vitamin C content than the unripe pepper,

the trend agrees with what Marin et al. (2004) reported for Capsicum annuum, L.cv.

Vergasa, and what Oboh et al. (2005) reported for Capsicum annuum, Tepin and

Capsicum chinese, Habanero. This goes a long way to confirm that ripe pepper will

likely be a better source of dietary vitamin C than unripe pepper. Vitamin C is found in

fruits, particularly fruits and juices and green leafy vegetables, they protect the body

against cancer of the oesophagus, oral cavity and stomach, it also helps to maintain the

blood vessel flexibility and improves blood circulation in the arteries of smokers as well

as facilitating the absorption of iron in the body. As an antioxidant, vitamin C’s primary

role is to neutralize free radicals; it can work for both inside and outside the cells to

combat free radical damage (Block et al. 1992; Umar et al. 1998).

However, Pietta (2000) reported that the phenolic compounds are responsible for

most of the antioxidant activity in plants; the presence of derivatives of cinnamic acid and

flavonoids has been found in pepper fruit. However, numerous studies have conclusively

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shown that the majority of the antioxidant activity may be from compounds such as

flavonoids, isoflavone, flavones, anthocyanin, catechin and isocatechin rather than from

vitamins C, E and β-carotene (Sun et al. 2002; Chu et al. 2002; Marin et al. 2004;

Materska & Perucka, 2005). In addition, to their role as antioxidant, some of them are

known to have anti-inflammatory and antiproliferative activities (Sun et al. 2002; Chu et

al. 2002). Polyphenols are capable of removing free radicals, chelate metal catalysts,

activate antioxidant enzymes, reduce α-tocopherol radicals, and inhibit oxidases (Amic et

al. 2003; Alia et al. 2003; Oboh, 2006a). The results of the total phenol content of the

pepper as shown in table 1 revealed that the ripe pepper had significantly higher (P<0.05)

total phenol content than the unripe pepper [ripe (117.7mg/100g) & unripe

(70.5mg/100g)]

However, the total phenol content of this pepper (ripe and unripe) is below that of

Capsicum annuum, Tepin, but within the same range with that of Capsicum chinese,

Habanero (Oboh et al. 2005). Furthermore, the trend in the phenol content with ripening

agrees with the trend in Capsicum chinese, Habanero (Oboh et al. 2005) and what

Materska and Perucka (2005) reported for some varieties of hot pepper [Capsicum annum

L (Bronowicka Ostra, Cyklon, Tomado and Tajfun)], in that the ripe pepper had higher

total phenol content than the unripe pepper. Furthermore, the total phenol content was

within the same range with what Chu et al. (2002) reported for broccoli, spinach, onion

and red pepper, but above that of carrot, cabbage, potato, lettuce, celery and cucumber.

As presented in figure 1 and table 2, aqueous extract of the ripe and unripe

Capsicum pubescens (1.7 – 8.3mg/ml) caused a significant decrease (P<0.05) in the lipid

peroxidation in the Rat’s brain homogenates in a dose-dependent manner. However, the

ripe pepper (EC50 = 3.5mg/ml) caused a significantly higher (P<0.05) inhibition in the

MDA production in the brain tissues than the unripe pepper (EC50 = 7.2mg/ml). The

inhibition of lipid peroxidation in the Rat’s brain by the aqueous extract could be ascribed

to the presence of both antioxidant vitamins and polyphenol compounds present in the

pepper. Furthermore, it is worth noting that ripening increases the ability of the pepper to

prevents MDA production in the brain tissue homogenates, unlike Capsicum annuum,

Tepin, that ripening decreases the protective ability of the pepper on brain tissue (Oboh et

al. 2005).

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This increasing in the protective ability of the aqueous extracts on the brain tissue

could be attributed to the fact that pepper undergoes some physiological changes during

ripening that leads to changes in the phytochemicals (Marin et al. 2004; Materska and

Perucka, 2005). This change in phytochemicals during ripening may have lead to an

increase in the total phenol and vitamin C content of the pepper. This may have

accounted for the enhanced protective ability of the ripe pepper, as many studies had

conclusively reported that there are correlation between plant phenol content and

antioxidant properties (Chu et al. 2002; Sun et al. 2002; Alia et al. 2003; Oboh, 2005b;

Oboh, 2006b). In addition, the EC50 of the pepper was higher than Capsicum annuum,

Tepin and Capsicum chinese, Habanero that are considered to be the hottest pepper

(Pepper Gallery, 2005)

Figure 2 shows the interaction (inhibition) of the pepper extract with Fe (II)-

induced lipid peroxidation in Rat’s brain. The result revealed that incubation of the brain

tissue in the presence of 25µM Fe (II) caused 260% increase in the MDA content of the

brain homogenates when compared with the basal brain homogenates. The increased lipid

peroxidation in the presence of Fe2+ could be attributed to the fact that Fe2+ can catalyze

one-electron transfer reactions that generate reactive oxygen species, such as the reactive

OH·, which is formed from H2O2 through the Fenton reaction. Iron also decomposes lipid

peroxides, thus generating peroxyl and alkoxyl radicals, which favors the propagation of

lipid oxidation (Zago et al. 2000). High levels Fe play a crucial role in brain cancer and in

degenerative diseases of the brain (Parkinson’s and Alzheimer's diseases, multiple

sclerosis, etc.) (Johnson, 2001).

However, aqueous extracts of Capsicum pubescens caused a significant inhibition

(P<0.05) in Fe (II) induced lipid peroxidation in the Rat’s brain in a dose-pedendent.

Nevertheless, the ripe pepper caused a significantly higher (P<0.05) inhibition in the

MDA production in the Rat’s brain tissue than the unripe pepper. The decrease in the

Fe2+ induced lipid peroxidation in the Rat’s brain homogenates in the presence of the

extracts could be as result of the ability of the extracts to chelate Fe2+. In addition,

scavenge free radicals produced by the Fe2+ catalyzed production of reactive oxygen

species (ROS) in the Rat’s brain homogenates by the antioxidant phytochemicals in the

extract of the peppers. Peppers are rich in antioxidants vitamin as well as phenols;

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phenolics compound are more potent antioxidant in foods than vitamin C and E (Marin

et al. 2004; Materska and Perucka, 2005; Oboh and Akindahunsi, 2004, Oboh, 2005a;

Oboh, 2006b). The higher ability of the ripe pepper to protect the rat’s brain could be

attributed to its higher phenol and vitamin C content.

The use of Fe chelator is a popular therapy for the management of Fe (II)

associated oxidative stress in brain. The Fe (II) chelating ability of the pepper extracts

are presented in figure 3. The results revealed that both extract significantly (P<0.05)

chelate Fe (II) in a dose-dependent manner. However, there was no significant (P>0.05)

difference in the Fe (II) chelating ability of the ripe and unripe pepper, nevertheless the

ripe pepper extract had higher Fe (II) chelating ability. It is worth noting that, correlation

exists between the total phenol content and Fe (II) chelating ability. Conversely, the

extracts did not significantly inhibits (P>0.05) OH radical (Fe (II) / H2O2) -induced

decomposition of deoxyribose as shown in figure 4. The reason for the inability of the

extract to prevent deoxyribose decomposition could not be categorically stated.

However, it is likely that the complex formed between the pepper phytochemicals and

Fe, could not initiate lipid peroxidation, but active in Fenton reaction.

Furthermore, the ripe pepper had a significantly higher (P<0.05) ability to reduce

Fe (III) to Fe (II) than the unripe pepper (figure 5). This result agrees with the trend in

vitamin C content presented in table 1. It could reason that phenol and vitamin C are

actively involved in the protection of brain tissue from Fe (II) induced lipid peroxidation,

the mechanism through they possibility do this is by their Fe(II) chelating ability and

reducing, and not through hydroxyl radical scavenging ability.

The interaction of the aqueous extract of Capsicum pubescens with sodium

nitroprusside and quinolinic acid induced lipid peroxidation in rat’s brain are presented

in figures 6 & 7 respectively. The results revealed that the extract caused a significant

decrease (P<0.05) in the brain MDA content in sodium nitroprusside and quinolinic acid

induced lipid peroxidation in a dose dependent manner. However, the ripe pepper had a

significantly higher (P<0.05) inhibitory effect in the lipid peroxidation induced by both

pro-oxidants than the unripe pepper. The higher inhibitory effect of the ripe pepper on

lipid peroxidation in the Rat’s brain is in line with the vitamin C and total phenol

content, reducing power and Fe chelating ability of the pepper.

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The protective properties of the pepper against sodium nitroprusside induced

lipid peroxidation on the brain could be because of the ability of the antioxidant

phytochemicals present in the aqueous extract to quench-scavenge the nitrous radical

and Fe produced from the decomposition of sodium nitroprusside. While the inhibition

of the quinolinic acid induced lipid peroxidation in the brain tissues could at least in part,

be by preventing over stimulation of NMDA receptor and free radical scavenging ability

of the phenol content.

CONCLUSION

Capsicum pubescens could prevent Fe (II), sodium nitroprusside and quinolinic acid

induced lipid peroxidation in brain, this they do through the antioxidant activity of their

phytochemicals (Phenols and vitamin C). However, ripening which brings about a

change in the pigment of pepper caused an increase in the phenol and vitamin C content;

and consequently cause an increase in the protective ability of the Capsicum pubescens

ability to protect rats brain from the pro-oxidant induced lipid peroxidation by enhancing

Fe (II) chelating and reducing power of the pepper.

ACKNOWLEDGMENTS

The authors wish to acknowledge the Conselho Nacional de Desenvolvimento Científico

e Tecnológico (CNPq) Brazil and Academy of Science for the Developing World

(TWAS), Trieste Italy; for granting Dr G. Oboh, Post-Doctoral fellowship tenable at

Biochemical Toxicology Unit of the Department of Chemistry, Federal University of

Santa Maria, Brazil. This study was also supported by CAPES, FIPE/UFSM, VITAE

Foundation and FAPERGS. In addition, the authors apppreciate the support of The Abdus

Salam International Centre for Theoretical Physics, Trieste, Italy. Financial support from

the Swedish International Development Cooperation Agency is acknowledged.

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Table 1: Total phenol content and Vitamin C content of Capsicum pubescens Sample Vitamin C Total Phenol (µg/g) (mg/100g gallic acid equivalent) Ripe 231.5±11.5a 117.7±10.5a Unripe 224.4±9.20a 70.5±7.8b Values represent means of triplicate. Values with the same alphabet along the same column are not significantly different (P>0.05)

Table 2: EC50 of Aqueous extract of Capsicum pubescensi Sample EC50 (mg/ml) Ripe 3.5 ±1.2 b Unripe 7.2±0.8a Values represent means of triplicate. Values with the same alphabet along the same column are not significantly different (P>0.05)

20

0

20

40

60

80

100

120

0 1,7 3,3 5 6,7 8,3

Concentration of extract (mg/ml)

MD

A (%

Con

trol)

Ripe pepper Unripe pepper

Figure 1: Capsicum pubescens inhibits lipid peroxidation in Rat´s brain.

200

210

220

230

240

250

260

270

280

0 4,2 8,4 12,6 16,8


MD

A (%

Con

trol)


Figure 2: Capsicum pubescens inhibits Fe (II) induced lipid peroxidation in Rat´s brain.

21

0

10

20

30

40

50

60

70

80

90

0 3,3 6,7 10 13,3 16,7


% F

e (II

) Che

latio

n


Figure 3: Fe (II) chelation ability of Capsicum pubescens.

0

20

40

60

80

100

120

0 4,2 8,4 12,6 16,8


% D

ecom

posi

tion

of D

eoxy

ribos

e


Figure 4: Inhibition of Deoxyribose Decomposition by Capsicum pubescens.

22

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0 3,3 6,7 10


OD

700


Figure 5: Reducing power of Capsicum pubescens.

0

20

40

60

80

100

120

140

160

180

200

0 4,2 8,4 12,6 16,8

Cocentration of extract (mg/ml)

MD

A (%

Con

trol)


Figure 6: Capsicum pubescens inhibits Sodium nitroprusside induced lipid peroxidation

in Rat´s brain.

23

0

50

100

150

200

250

0 4,2 8,4 12,6 16,8


MD

A (%

Con

trol)


Figure 7: Capsicum pubescens inhibits Quinolinic acid induced lipid peroxidation in

Rat´s brain.

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