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Annals of Agricultural Science (2012) 57(2), 167–173
Faculty of Agriculture, Ain Shams University
Annals of Agricultural Science
www.elsevier.com/locate/aoas
Original Article
Antiatherogenic properties of vegetable juice rich
in antioxidants in cholesterol-fed rats
G.A.T.A. El-Shatanovi a, I.S. Ashoush a,*, Enaam K. Ahmed b, Soad A. Ali c
a Food Sci. Dept., Fac. of Agric., Ain Shams Univ., Shoubra El-Kheima, Cairo, Egyptb National Organization for Drug Control Research, Giza, Egyptc Cairo University Hospitals, Kasr El-Aini St., Cairo, Egypt
Received 5 February 2012; accepted 23 February 2012Available online 25 October 2012
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KEYWORDS
Vegetable juice;
Antioxidants;
Antiatherogenic;
Rats
Corresponding author. Mo-mail address: ihab.ashoush
er review under responsibilit
niversity.
Production an
70-1783 ª 2012 Faculty of A
tp://dx.doi.org/10.1016/j.aoas
bile: [email protected]
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gricultur
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Abstract Vegetable juice was prepared from a mixture of tomato, watercress, parsley, carrots, cel-
ery, lettuce, beets and spinach and analyzed for its antioxidant content and activity (total phenols,
total flavonoids, carotenoids, chlorophyll A and B, vitamins C and E) and minerals. As well as anti-
atherogenic effects of vegetable juice on Wister rats fed on high fat diet was studied. The results
revealed that, the vegetable juice had a high content of important compounds which act as antiox-
idants e.g., phenolic compounds (22.37 mg/L), lycopene (84.24 mg/100 g), vitamin C (5.25 mg/mL),
carotenoids (0.93 mg/L) and Zn (5.02 mg/100 g). The biological evaluation indicated that, the rat’s
body weight gain was significantly higher in the atherogenic control group (Ac) than those of the
other two groups [normal control group (NC) fed on standard diet and vegetable juice group
(VJ) fed on atherogenic diet and consumed vegetable juice (1 mL/g BW)]. An improvement in serum
lipid profile of VJ group was observed. The histopathological examination of the heart and aorta
confirmed these results which reflected the protective effect role of vegetable juice against athero-
sclerosis and cardiovascular diseases.ª 2012 Faculty of Agriculture, Ain Shams University. Production and hosting by Elsevier B.V. All rights
reserved.
Introduction
Cardiovascular disease is caused by disorders of the heart andblood vessels, and includes coronary heart disease (heart at-
tacks), cerebrovascular disease (stroke), raised blood pressure
01001843122.om (I.S. Ashoush).
lty of Agriculture, Ain-Shams
g by Elsevier
e, Ain Shams University. Producti
.011
(hypertension), peripheral artery disease, rheumatic heart dis-
ease, congenital heart disease and heart failure. The majorcauses of cardiovascular disease are tobacco use, physical inac-tivity, and an unhealthy diet. Cardiovascular diseases are theworld’s largest killers, claiming 17.1 million lives a year
(WHO, 2011). Cardiovascular disease, including coronaryheart disease, stroke, and peripheral vascular disease, is theclinical expression of advanced atherosclerosis. Variation in
atherosclerosis between individuals can be partially explainedby a collection of ‘‘risk factors’’, with serum cholesterol con-centration, distribution of cholesterol among lipoproteins,
blood pressure, and smoking status contributing significantlyto risk of atherosclerosis-related diseases (Dawn, 1998). In
on and hosting by Elsevier B.V. All rights reserved.
168 G.A.T.A. El-Shatanovi et al.
recent years, increasing attention has been paid to the role of
diet in human health. Several epidemiological studies haveindicated that a high intake of plant products is associatedwith a reduced risk of a number of chronic diseases, such asatherosclerosis and cancer (Gosslau and Chen, 2004; Gundg-
aard et al., 2003). These beneficial effects have been partlyattributed to the compounds which possess antioxidant activ-ity. According to the US Food and Drug Administration
(FDA) antioxidants are defined as ‘‘substances used to pre-serve food by retarding deterioration, rancidity, or discolor-ation due to oxidation’’. Antioxidant also was defined with a
broader perspective as ‘‘any substances when present at lowconcentration compared to those of an oxidizable substrate,significantly delays or prevents oxidation of the substrate’’.
The major antioxidants of vegetables are vitamins C and E,carotenoids, and phenolic compounds, especially flavonoids.Parvin et al. (2009) demonstrated that higher consumption offruits and vegetables has been found to be potentially protec-
tive against a cardiovascular disease. Sprecher and Pearce(2002) demonstrated that diets rich in fruits and vegetablesare good sources of dietary fiber. In a randomized trial, a fi-
ber-multivitamin combination resulted in a reduction inLDL–C of 8% from baseline after 8 weeks.
The present study was designed to assess the antiatherogen-
ic effects of vegetable juice consumption on the lipid profileand histopathological alteration in rats fed high fat diet. Theantioxidant activity and vegetable juice contents were alsoinvestigated.
Materials and methods
Materials
Vegetables used throughout this work included; tomato,watercress, parsley, carrots, celery, lettuce, beets and spinachobtained from the local market, Cairo, Egypt. Commercial kits
used for determining serum lipid parameters were purchasedfrom Biodiagnostic Co., Dokki, Egypt. 1,1-diphenyl-2-pic-rylhydrazyl radical (DPPH) and Folin–Ciocalteus phenol re-
agent was purchased from Sigma–Aldrich Inc. (St. Louis,MO, USA).
Preparation of vegetable juice
The fresh vegetables were purchased and immediately withoutstorage, squeezed to juices. The edible portions of deep-colored
vegetables were weighed, washed and chopped, respectively, tosqueeze vegetable juice using a manual stainless screw squeezer(Vegetable Grinder). The juices were centrifuged at 10,000g at
4 �C for 30 min, then the supernatants were collected using suc-tion filtration through filter papers (Toyo No. 5B) and blindedwith ratio to produce blinding vegetable juice stored at �20 �Cfor chemical analyses and biological experiment.
Chemical composition of vegetable juice
Moisture content, crude protein, lipids, crude fiber and ashwere determined according to the method of the A.O.A.C(2007). Total dietary fiber, soluble and insoluble dietary fibers
were determined according to the method described by Proskyet al. (1988).
Determination of antioxidant content
Total phenolic in vegetable juice was determined by folin–ciocalteau’s reagent as described by Arnous et al. (2001). The
amount of total flavonoid content was determined by alumi-num chloride method of Chang et al. (2002). Chlorophyll-A,chlorophyll-B and carotenoids were extracted and calculated
according to the method of Litchenthaler (1987). Lycopenepigments were determined according to the method describedby Sadler et al. (1990). Vitamins C and E were determinedusing HPLC (Hewlett Packard series 1100) according to Zhao
et al. (2004).Mineral elements of Mn, Zn, Na, and K were determined
according to the method described in A.O.A.C (2007) using
atomic absorption spectrophotometer.
Evaluation of the free–radical scavenging capacity
The ability of vegetable juice to scavenge DPPH free radicalswas determined by the method described by Lim et al.
(2001). The percent of antiradical activity (ARA) againstDPPH was calculated according to the following equation:
ARA% ¼ ½ðabsorbance of control� absorbance of sampleÞ=absorbance of control� � 100:
Sensory evaluation of vegetable juice
Ten members semi trained panel from the staff of Food ScienceDepartment Ain Shams University were asked to score the pre-
pared vegetable juice compared to a commercial one for theirappearance, color, taste, odors, consistency and overall accept-able; giving numerical scores to each of their attributes from 10,using a report sheet according to Watts et al. (1989).
Biological experiment
The experiment was conducted on 24 male Wistar rats with anaverage weight of (140 ± 10 g). The rats were obtained fromthe Organization of Biological Products and Vaccines (Helwan
Farm, Cairo, Egypt). The rats were housed in screen-bottomedaluminum cages in rooms maintained at 25 ± 1 �C with alter-nating cycles of light and dark for 12 h duration. The rats werefed on the control diet for seven consecutive days. Rats were
randomly divided into three groups, each of eight rats, the firstgroup was normal control (NC) fed on the normal control diet,the second was atherogenic control (AC) group fed on athero-
genic diet and the third was vegetable juice group (VJ) fed onatherogenic diet and given orally vegetable juice in a dose of1 mL/100 g body weight per day for successive 10 weeks. The
calculation was based on a consumption of 275 mL/day for a70 kg human as reported by Rouanet et al. (2010). The compo-sition of the experimental diets is shown in Table 1 according
to AIN-93 guidelines (Reeves et al., 1993).The changes in body weight were recorded weekly. Blood
samples were withdrawn from the retro-orbital plexus of theeyes from each rat according to the procedure of Schermer
(1967) at time intervals 0, 15, 30, 45, 60 and 75 days of theexperiment; serum was separated by centrifugation at1500 rpm for 15 min at ambient temperature to estimate the
biochemical parameters.
Table 1 Compositions of the experimental diets (%).
Composition (%) Normal diet Atherogenic diet
Corn starch 60.5 59.2
Casein 20 20
Corn oil 10 –
Cellulose 5 5
Salt mixture 3.5 3.5
Vitamin mixture 1 1
Cholic acid – 0.3
Cholesterol – 1
Lard – 10
Table 2 Chemical composition of vegetable juice.
Parameters (%)
Moisture 90.52 ± 0.06
Ash 2.48 ± 0.01
Fat 2.30 ± 0.10
Protein 1.20 ± 0.10
Crude fiber 3.50 ± 0.10
Total dietary fiber 6.05 ± 0.15
Soluble dietary fiber 4.14 ± 0.04
Insoluble dietary fiber 1.94 ± 0.11
Data are the mean ± SD, n= 3.
Table 3 Antioxidant contents and activity of vegetable juice.
Components Concentration
Total phenol (mg/L) 22.37
Total flavonoids (mg/L) 0.96
Carotenoids (mg/L) 0.93
Total chlorophyll (mg/L) 3.09
Chlorophyll-A 1.36
Chlorophyll-B 1.27
Lycopene (mg/100 g) 84.24
Vitamin C (mg/mL) 5.25
Vitamin E (mg/mL) 1.33
Scavenging activity (%) 94.37
Table 4 Some minerals content of vegetable juice (mg/100 g).
Minerals Concentration (mg/100 g)
Mn 3.97
Zn 5.02
Na 197.73
K 92.42
Antiatherogenic properties of vegetable juice richin antioxidants in cholesterol-fed rats 169
Lipid profile parameters
Enzymatic determination of total cholesterol (TC) in serum
was carried out according to Roeschlau et al. (1974). Full enzy-matic determination of total triglycerides (TG) in serum wascarried out calorimetrically at 546 nm, according to Fossatiand Prencipe, 1982, low density lipoproteins–cholesterol
(LDL–C) was determined by enzymatic methods of Wielandand Seidel (1983). While, the high density lipoproteins–choles-terol (HDL–C) was determined according to the method of
Lopez-Virella et al. (1977).
Histopathology
Autopsy samples were taken from the heart and aorta of ratsin the different groups and fixed in 10% formol saline for 24 h.
Washing was done in tap water then serial dilutions of alcohol(methyl, ethyl and absolute ethyl) were used for dehydration.Specimens were cleared in xylene and embedded in paraffin
at 56� in a hot air oven for 24 h. Paraffin bees wax tissue blocks
were prepared for sectioning at 4 lm by slidge microtome. Theobtained tissue sections were collected on glass slides,deparaffinized and stained by hematoxylin and eosin stains(Banchroft et al., 1996) for histopathological examination
through the electric light microscope.
Statistical analysis procedure
Data of antioxidant content and activity, as well as sensoryevaluation were expressed as mean values ± SD. Statistical
analysis was performed using one way analysis of variance(ANOVA) followed by Duncan’s Multiple Range Test withP < 0.05 being considered statistically significant. Data for
biological study were exposed proper statistical analysis of var-iance for repeated measurements using SAS program (SAS,1996).
Results and discussion
Chemical composition of vegetable juice
The results of chemical analysis of vegetable juice presented in
Table 2 showed that vegetable juice contained a high amountof moisture and a low amount of protein. The total dietary fi-ber content was 6.05% and the soluble fiber was found to be
higher than the insoluble fiber.
Antioxidant contents and activity of vegetable juice
The data in Table 3 summarized the antioxidant componentsof vegetable juice showed that the juice contained a highamount of total phenols, lycopene and vitamin C (22.37 mg/
L, 84.24 mg/100 g and 5.25 mg/mL, respectively). On the otherside, the vegetable juice had a moderate content of total chlo-rophyll (3.09 mg/L), while it contained low amounts of total
flavonoids and carotenoids (0.96 and 0.93 mg/g respectively).Furthermore, vitamin E content was 1.33 mg/mL. These re-sults are in agreement with those of Paganga et al. (1999)
and Proteggente et al. (2002).For scavenging activity of vegetable juice (Table 3), the data
revealed that vegetable juice had much free radical scavengingactivity. Therefore, this juice increases health benefits by
increasing antioxidant properties and prevention of oxidationdamage as reported by Cao et al. (1998) and Bazzano (2006).
From Table 4, it could be observed that vegetable juice had
an abundant content of the macroelement Na (197.73 mg/100 g) and K (92.42 mg/100 g), while the other microelementscontent arranged in ascending order as follows: Zn (5.02 mg/
100 g) and Mn (3.97 mg/100 g). Generally, it is well knownthat minerals Zn and Mn are active as antioxidant accordingto Gorinstein et al. (2011).
Table 6 Effect of oral administration of vegetable juice on body weight gain in rats.
Animal group Initial body weight (g) Final body weight (g) Weight gain (g)
NC 144.2 ± 7.88a 169.65 ± 24.98b 25.47 ± 19.69b
AC 146.3 ± 5.81a 194.57 ± 23.41a 48.30 ± 20.06a
VJ 142.7 ± 11.13a 142.7 ± 11.13c 25.77 ± 8.96b
Means having different letters (superscript) in the same column are significantly different (P < 0.05).
0
20
40
60
80
100
120
140
160
180
0 15 30 45 60 75
Tri
gly
ceri
de (
mg/
dl)
Time course (days)
NC
AC
VJ
0
20
40
60
80
100
120
140
160
180
200
0 15 30 45 60 75
T.
Cho
lest
erol
(m
g/dl
)
Time course (days)
NC
AC
VJ
0
10
20
30
40
50
60
70
80
0 15 30 45 60 75
HD
L (
mg/
dl)
Time course (days)
NC
AC
VJ
0
20
40
60
80
100
120
140
0 15 30 45 60 75
LD
L (
mg/
dl)
Time course (days)
NC
AC
VJ
0
1
2
3
4
5
6
7
8
0 15 30 45 60 75
Ris
k ra
tio
(%)
Time course (days)
NC
AC
VJ
0
1
2
3
4
5
6
7
0 15 30 45 60 75
Ath
erog
enic
Ind
ex (
%)
Time course (days)
NC
AC
VJ
Fig. 1 Effect of oral administration of vegetable juice on serum lipids profile in rats. (The data are presented as means ± SD from eight
rats per group in 10 week-periods.)
Table 5 Sensory characteristics of vegetable juice.
Juices Appearance Color Taste Odors Consistency Overall
Prepared vegetable juice 9.0a 9.4a 8.8a 8.1a 8.3a 42.8a
Commercial vegetable juice 7.9b 7.9b 7.4b 7.7a 7.9a 39.6a
Means having different letters (superscript) in the same column are significantly different (P < 0.05).
170 G.A.T.A. El-Shatanovi et al.
Sensory characteristics of vegetable juice
The mean score values for all parameters of sensory evaluation
in fresh vegetable juice including: appearance, color, taste,odors, consistency and overall acceptable are represented in
Table 5. Non-significant differences were noticed between juicesin the odors and consistency characteristic, while, significantdifferences were found in the appearance, color and taste. Theseresults confirm the suitability of vegetable juice in the produc-
tion of healthy and simply applicable form in the human diet.
Fig. 2 Heart of a normal control rat showing the normal
histological structure of myocardium (H & E, 160·).
Fig. 3 Aorta of a normal control rat showing the normal
histological structure of the intima and media (H & E, 64·).
Fig. 4 Heart of atherogenic rat showing wide depression in
myocardium (H & E, 160·).
Fig. 5 Aorta of atherogenic rat showing vacuolization in the
tunica media (H & E, 160·).
Fig. 6 Heart of rat treated with vegetable juice showing
depression in the bundles of the myocardium (H & E, 160·).
Antiatherogenic properties of vegetable juice richin antioxidants in cholesterol-fed rats 171
Biological evaluation
Effect of vegetable juice on body weight of ratsResults in Table 6 showed that after 10 weeks of feeding; bodyweights were significantly higher in rats group fed on athero-genic diet compared to other groups. It was clear that weight
gain was influenced by the type of dietary fat in the diet. Itseems that the lipids were accumulated in rats’ bodies fed onatherogenic diet without consumption of vegetable juice, whilerats fed on the same atherogenic diet in addition to vegetable
juice showed a significant less weight gain and statisticallyequal to those fed on normal control diet.
Lipid profile parametersConcentrations of serum lipid profile are shown in (Fig. 1).The concentration of total cholesterol (TC) and LDL–C in ser-
um of both rat groups fed on atherogenic diet was greater thanrats fed on normal diet. TC and LDL–C were significantlygreater in rats fed atherogenic diet than those consumed vege-
table juice. Therefore, the vegetable juice significantly reducedserum TC concentration by 25.84% and LDL–C by 96.9%.These findings are in agreement with those obtained by Brown
et al. (1999) and Agarwal and Rao (1998).The greater concentration of TG in rats fed on atherogenic
diet than those in the other groups indicated that the vegetablejuice significantly prevented the rise of serum TG in rats fed on
high fat diet (79.99% vs. atherogenic group at the end ofexperiment).
Total cholesterol/HDL ratio increased significantly in rats
fed on atherogenic diet while it statistically decreased in ratsconsumed vegetable juice and control group. For this reason,
Fig. 7 Aorta of rat treated with vegetable juice showing the mild
vacuolization in the tunica media (H & E, 64·).
172 G.A.T.A. El-Shatanovi et al.
atherogenic index (AI) was higher significantly in the athero-genic group than in the NC and VJ consumed groups.
The risk ratio (total cholesterol/HDL–C) and atherogenic
index [(total cholesterol � HDL–cholesterol)/HDL–C] are alsopredictors of coronary risk. A significant increase in risk ratioand atherogenic index was observed in atherogenic rats that
has an effect on cardiovascular diseases. Vegetable juice mayplay an important protective role against atherosclerosis andcardiovascular disease in mammals, which explained thereduction of these ratios in rats group, consumed vegetable
juice.
Histopathological examinationData in Figs. 2–7 reveal the histopathological examination ofsemi-thin sections of the heart and aorta of rats stained withhematoxylin and eosin. The group fed on normal control diet
(NC) had no histopathological alteration in the myocardium(m), Fig. 2; as well as in the tunica intima (i), media (m) andtunica adventitia (a) of the aorta Fig. 3. Treatment with
atherogenic control group (AC) caused wide depression in be-tween the myocardial bundles, Fig. 4. There was vacuolizationin the tunica media of the aorta Fig. 5.
Oral consumption of vegetable juice of rats fed on athero-genic diet had depression in the bundles of the myocardium,Fig. 6. Mild vacuolization was observed in the tunica mediaof the aorta, Fig. 7. These findings are in harmony with those
obtained by Bansal et al. (2002) who reported that atheroscle-rotic lesions are significantly more developed in animalexperimental group fed on high fat diet than other groups.
Therefore, the previous results of the histopathological exam-ination confirmed those of the biological evaluation in thepresent investigation which justify that the consumption of
vegetable juice reflects a protective effect role against athero-sclerosis and cardiovascular diseases.
Conclusion
Based on the above results, it could be concluded that the veg-
etable juice could be used as a source of dietary antioxidantsincluded, bioactive plant phenols, and flavonoids. The healthbenefits of vegetable juice are largely due to the antioxidantcontents, dietary fiber and vitamins supported by the large
number of phytochemicals due to significantly hindered in-
creases in TC, TG, LDL–C and atherogenic index levels.Therefore, the antiatherogenic effect of vegetable juice shouldbe investigated in the human diet for application in preventingatherosclerosis and cardiovascular disease.
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