Basic Research Journal of Medicine and Clinical Sciences ISSN 2315-6864 Vol. 2(11) pp.

116-121 December 2013

Available online http//www.basicresearchjournals.org
Copyright 2013 Basic Research Journal


Full Length Research Paper


The effects of red guava (Psidium guajava L) fruits on
lipid peroxidation in hypercholesterolemic rats

Sugeng Maryanto

Nutrition Science Study Program, School of Health Ngudi Waluyo. Jl. Gedongsongo-Candirejo, Ungaran-Semarang-
Indonesia

*Corresponding author email: sugengmaryanto99@yahoo.co.id

Accepted 10 December, 2013

Background: Lipid peroxidation is a mechanism of cell injury, characterized by the formation of free
radicals including malondialdehyde (MDA). To prevent or reduce the injury by free radicals,
antioxidants are needed. Red Guava Fruit (Psidium guajava L) contains high antioxidants including
vitamin C. This study aimed to determine the effects of red guava fruit on lipid peroxidation in
hypercholesterolemic rats. Method: This study, a randomized Pre test-Post test control group design,
was done on hypercholesterolemic Sprague Dawley rats. The rats were treated with 0.72 g red guava
flour and vitamin C supplements equal to that in the red guava flour as a comparison for 14 days. SOD
was measured by using the reaction of xanthine and xanthin oxidase (XOD). MDA was measured with
tiobarbiturat acid (TBA) reaction. Data were analyzed by using a t-test and one way Anova with 5% level
of significant. Result: There were an increase of SOD and decrease of MDA at both groups at almost the
same percentage, 73% to 74% and 61% to 62% respectively. The mean of SOD and MDA post
supplementation values of guava and vitamin C groups were almost at the same level with the group
receiving standard diet, which were significantly higher and lower than the high cholesterol diet group
respectively. Conclusion: Red guava fruit has the same antioxidant potency as vitamin C.

Keywords: red guava fruit, SOD, MDA, lipid peroxidation.


INTRODUCTION

Lipid peroxidation, a mechanism of cell injury, is
characterized by unstable free radical formation and is
decomposed to form some complex compounds including
reactive carbonyl compounds, mainly malondialdehyde
(MDA). MDA is a highly reactive compound that is the
end product of lipid peroxidation and is usually used as a
biomarker for assessing lipid peroxidation of biological
oxidatife stress (Guraud, 2010).

To prevent or reduce chronic diseases by free radicals,
antioxidants are needed. Antioxidants stabilize free
radicals by completing the deficiency of electrons
possessed by free radicals, inhibiting the chain reaction
and the formation of free radicals that can cause
oxidative stress. There are two types of antioxidants that
are exogenous and endogenous antioxidants. Exogenous
antioxidants are material from outside the body, entering
through food or supplements. Food included in the
exogenous antioxidants are: Alpha lipoic acid (ALA),
Bilberry (Vaccinium myrtillus), Burdock (Artium lappa),
Carotenoids, Coenzyme Q 10, Curcumin (Tumeric),
Flavonoids, Garlic, Ginkgo biloba, Glutathione, Grape
seed extract, Green tea, Melatonin, methionine, N-






Acetylcysteine (NAC), Nicotinamide Adenine dinucleotide
(NADH), oligomeric Proanthocyanidins (OPCs),
Pycnogenol, Selenium, Silymarin, Vitamin A, Vitamin C,
Vitamin E and Zinc. While endogenous antioxidant are a
natural antioxidant enzymes in the human body which are
superoxide dismutase (SOD), glutathione peroxidase
(GPx), and katalase (Bender, 2009).

Both supplement and antioxidant intake in the diet can
increase the synthesis of endogenous antioxidant
enzymes in the blood such as SOD (superoxide
dismutase), GPx (glutathione peroxidase) and catalase
(Castilla et al., 2008; Herrera et al., 2009).

The presence
of endogenous antioxidant enzymes in the blood plays a
role in the inhibition of atherosclerosis by preventing the
oxidation of LDL, in addition, to the endogenous
antioxidants can reduce lipid peroxidation. There were
clinical studies mentioning that the daily consumption of
antioxidants in the diet can reduce the risk of
atherosclerosis due to the oxidation of LDL and can
prevent premature vascular diseases (Mukherjee and
Das, 2011).
Fruits and vegetables contain high ascorbic acid, -
carotene and folic acid which are useful to the body as a
bioactive that is directly related to the prevention of lipid
peroxidation and vascular disease (Farinazzi-Machado et
al., 2012).

Guava (Psidium guajava L) wich is rich with
antioxidant is a tropical plant from America, spread to
Southeast Asia, including Indonesia. Several types of
guava are available, one of the most favored by
Indonesia people is red guava (Ditjen BPPHP
Departemen Pertanian, 2002).
Guava plants have many benefits. The leaves are used
traditionally by many people to overcome a wide range of
diseases, while the guava fruits are believed to overcome
various of health problems including and a source of
antioxidants (Sandra et al., 2012; Gutirrez et al, 2008).
Red guava fruit is also believed to help cure dengue
hemorrhagic fever (DHF) (Ditjen BPPHP Departemen
Pertanian, 2002). Guava fruit contains vitamin C, two
times higher than other fruits such as orange
(Mahattanatawee et al., 2006; Lim et al., 2006). Vitamin
C is an important compounds that has an antioxidant
activity (Thaipong et al., 2005; Monrrez-Espino et al.,
2011; Thuaytong and Anprung, 2011). Other compounds
in guava fruit are carotenoids such as beta-carotene,
lycopene, and beta-cryptoxanthin, and polyphenols
(Nascimento et al., 2010; Oliveira et al., 2010; Ordez-
Santos and Vzquez-Riascos, 2010). Lycopene is
associated with the prevention of cardiovascular damage
due the LDL oxidation, as the impact of dyslipidemia
(Lorenz et al., 2012; Sesso, 2012).
Several studies about the benefits of guava have been


Sugeng. 117



done, either using guava leaves or the fruit. The research
using guava leaves are proven to be beneficial for health,
such as for cough medicine, antidiabetic, antimicrobes,
antihypertensives, anti-inflammatory, analgesic,
antidiarrhea and spermatoprotective (Jaiarj et al., 1999;
Oh et al., 2005; Deguchi and Miyazaki, 2010; Qadan et
al., 2005; Ojewole, 2005; Ojewole, 2006; Akinola, 2007;
Gonalves et al., 2008).

Several studies have proven the
benefits of guava fruit for health. Antioxidant content in
guava fruit can be used as a source of natural
antioxidants (Jimnez-Escrig, 2001).

Lycopene content in
guava fruit is proven to prevent or delay the oxidative
damage of lipids, proteins and nucleic acids caused by
reactive oxygen species (ROS) (Lim et al., 2006).

This study aimed to determine the effects of red guava
fruit on lipid peroxidation in hypercholesterolemic rats.
The expected benefit is the find of scientific explanation
on the potency of red guava fruit in lowering lipid
peroxidation as a contributing factor to atherosclerosis.


METHODS

Experimental design

The study was an experimental design with randomized
Pre test-Post test control group design (Campbell and
Stanley, 1966). The minimum sample size of each group
was calculated using Federer formula, and was set out a
number of 7 animals in each group (Federer, 1991).


MATERIALS

Red guava fruit in this study was the ripe fruit from the
tree plantation in Sukorejo district Kendal regency,
Central Java, Indonesia. Guava pulp was dried in an
oven at a temperature of 60 C to be used as an animal
experiment food. Vitamin C content in the flour was
analyzed by using AOAC titrimetric method (Ball, 2006).
The content of vitamin C in red guava fruit flour was
0.398 mg/g.


Animal care and maintenance

Twenty-eight male Sprague Dawley rats aged 2 months
weighing range of 150 -160 g from integrated research
and testing laboratories (LPPT) Gadjah Mada University,
were divided into 4 groups, fed with the American
Institue of Nutition Rodent Diets (AIN 93) standard for 3
days, followed by a high-cholesterol food of AIN 93




118. Basic Res. J. Med. Clin. Sci.


Table 1. SOD levels in the various groups

Groups Means (U/gHb) T-test
Pre test SD Post test SD (%) (p-value)
Normal food (N) 688.9 50.31
a
687.7 34.74
a
-1,2 (-0.2) < 0.092
High Cholesterol food (C) 372.9 29.76
b
403.2 20.67
b
30.3 (8) < 0.062
Guava group (E1) 364.4 19.57
b
631.3 20.43
c
266.9 (73) < 0.001
Vitamin C group (E2) 368.0 24.19
b
639.2 20.34
c
271.2 (74) < 0.001

Note: The number followed by the same superscript letters indicate no difference (Anova)


Table 2. MDA levels in the various groups

Groups Means (mmol/L) T-test
Pre test SD Post test SD (%) (p-value)
Normal food (N) 1.89 0.31
a
2.02 0,30
a
0.13 (7) < 0.001
High Cholesterol food (C) 5.98 0.32
b
6.23 0.29
b
0.25 (4) < 0.002
Guava group (E1) 6.69 0.30
c
2.64 0.26
c
-4.05 (-61) < 0.001
Vitamin C group (E2) 7.21 0.34
d
2.74 3.35
c
-4.47 (-62) < 0.001

Note: The number followed by the same superscript letters indicate no difference (Anova)


standard (by adding 1% cholesterol crystals and sodium
cholate) for 14 days (Reeves, 1997). After doing
randomization, they were clustered in individual cages
and continued the treatment for 28 days. First group
received normal food (N) and the control group (C) was
given high-cholesterol only; treatment group I (E
1
)
received high-cholesterol food plus red guava fruit; and
treatment group II (E
2
) received vitamin C equal to the
content in red guava flour. Red guava fruit flour and
vitamin C treatment were calculated as an adult dose
converted based on Laurence and Bacharach to 200g
rats (Ngatijan, 2006).


Collection of blood samples and determination of
biochemical profiles

Blood samples were taken using a hematocrit pipette
through retro-orbital sinus in 1 ml to measure the levels of
Superoxide dismutase (SOD) and Malondialdehyde
(MDA) were performed before and after supplementation.
(Suckow et al., 2000). SOD was measured based on the
reaction of xanthine and xanthin oxidase (XOD) to
generate superoxid radicals that react with 2-(4-
iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium
chloride (INT) superoxid formazan dye which produces
one unit of SOD inhibits 50% reduction of INT
(Matsunami et al., 2010).

MDA was measured with the
acid-base reaction tiobarbiturat MDA (TBA) which form
colored compounds MDA-TBA2 and absorb light at a
wavelength of 532-534 nm, using spectrophotometer
(Tkzkan et al., 2006).

Statistical analysis

The variables are presented as means and standard
deviations. The data were analyzed by T-test and LSD-
test with 5% level of significance.
The study was conducted after obtaining the Ethical
Clearance from the Health Research Ethics Committee of
Diponegoro University and Dr. Kariadi Hospital.


RESULT

SOD and MDA profiles

Table 1 and 2 depict the level of SOD and MDA before
and after the supplementation.
There were significant increase in SOD level in groups
receiving supplementation up to 73%-74%. While in
MDA, the supplementation resulted the decrease value in
both groups by 61%-62%.
The Pre test mean value of the groups given high
cholesterol food had lower SOD values, half of the group
receiving normal food. However, with vitamin C and
guava supplementation both groups had higher SOD
level than the group receiving high cholesterol food with
no supplementation.
The outcome of the MDA level show that at the pre
supplementation, the normal food group had the lowest
level value, and the all three groups receiving high
cholesterol food had MDA level more than three times
higher. At the end of the study of MDA level, the groups
receiving guava and vitamin C had the same value,
almost half of the group receiving high cholesterol food


Sugeng. 119



Table 3. SOD and MDA in different test groups






Note: The number that followed by the same superscript letters
indicate no difference


only. Table 3 shows that guava fruit and vitamin C
supplementation give the same result in term of
increasing the MDA level but gives different result in
lowering the MDA level.


DISCUSSION

The study proves that red guava fruit increases levels of
SOD significantly at p <0.001 (Table 1). Different test
results between the groups showen that there were no
differences between groups E
1
(guava) and E
2
(vitamin
C) (Table 2). Based on these results it is said that red
guava fruit has the similar to vitamin C which increases
the levels of SOD in hypercholesterolemic rats. Guava
fruit is a natural source of exogenous antioxidants
including vitamin A, Phenols and Ascorbic Acid (Vitamin
C) (Lim et al., 2006). The results of this study indicate
that the consumption of guava fruit can increase
endogenous antioxidant status, which is characterized by
increased levels of SOD. The results are consistent with
the research conducted by Hermsdorff of dietary
antioxidants. It was reported that eating foods that
contain high antioxidants (exogenous antioxidants) may
increase the endogenous antioxidant status (enzymes
SOD, GPx and catalse) (Hermsdorff, 2011).

SOD is produced naturally by the body, but the amount
depends on the intake of exogenous antioxidants as well
as the state of one's body as well as the amount of free
radicals in the body. The normal or balance amount of
free radicals is important for the health and normal
function of the normal to fight inflammation, kill bacteria,
and control smooth muscle tone of blood vessels and
organs in the body. In an excessive / unbalanced
condition, free radical molecules wich are not stable in
the body are transformed into prey molecules, they will
begin to move wildly and attack healthy or unhealthy
body, causing disease. SOD is an endogenous
antioxidant enzyme of glutathione peroxidase (GPx) and
catalase, playing a role in counteracting free radicals in
the body (Bender, 2009).

The presence of antioxidants is associated with a
reduced risk of cardiovascular diseases, this is because
the principle mechanism of antioxidants inhibits LDL
oxidation and protects against nitric oxide. To repair this
situation, it needs an exterminator of free radicals known
as antioxidants. Free radicals in the blood vessels are
associated with the oxidation of LDL, as the beginning of
atherosclerosis. Knekt et al in their study explained
that consumption of antioxidants can suppress the
development of atherosclerosis by reducing oxidation of
LDL to LDL-ox (Knekt et al., 2004).

It was also reported
by Plotnick et al in Katz that supplementation of vitamin C
and E can prevent endothelial dysfunction, thereby
potential as cardioprotective (Katz and Friedman, 2008).


Besides being able to enhance endogenous antioxidant
SOD, red guava fruit consumption may reduce lipid
peroxidation, which is characterized by decreased levels
of MDA. This can be seen in Table 2, the red guava fruit
(E
1
) showed to decrease the MDA hypercholesterolemic
rats significantly (p <0.001). MDA is one of several lipid
molecules as the result of decomposing endoperoksida
formed during the process of lipid peroxidation, MDA,
therefore, is very appropriate to be used as an indicator
of lipid peroxidation (Guraud, 2010).

Lipid peroxidation
depends on the lipid intake and exogenous antioxidants
from food and lioid level in blood. Knekt et al in their
study of antioxidant vitamins and risk of CHD reported
that antioxidants play a role in lipid peroxidation in the
form LDL-ox. The resistance of antioxidant against lipid
peroxidation may inhibit or prevent the formation of MDA
which is characterized by reduced MDA levels (Knekt et
al., 2004). The results also showed that red guava fruit
was proven to lower LDL cholesterol significantly. Thus,
LDL peroxidation in the form of formed LDL-ox is also
reduced, so the MDA formed is reduced.
Based on the discussion, it was confirmed that the red
guava fruit as a source of exogenous antioxidants is able
Parameter Groups () means SD
1. SOD N -1,21 66,09
a

C 30,3 34,89
b

E1 266,9 26,54
c

E2 271,2 35,28
c

2. MDA N 0,013 0,06
a
C 0,3 0,13
b
E1 -4,1 0,23
c
E2 -4,5 0,39
c


120. Basic Res. J. Med. Clin. Sci.



to reduce lipid peroxidation shown by the increase of
endogenous SOD and the decrease of MDA level. Based
on the results of this study, a further study of red guava
fruit supplementation/consumption in human can be
considered.


CONCLUSSION

Red guava fruit is able to reduce lipid peroxidation
showed by the increase of SOD and the decrease of
MDA levels in animal experiment.


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