Content uploaded by Nora El-Sheikh
Author content
All content in this area was uploaded by Nora El-Sheikh on Jul 06, 2017
Content may be subject to copyright.
Australian Journal of Basic and Applied Sciences, 5(8): 1344-1353, 2011
ISSN 1991-8178
Corresponding Author: Fatma H. Abd El-Razek, Biochemistry and Nutrition Department, Women's College-Ain-Shams
University
E-mail: fattmahassan@yahoo.com
1344
Effects of Dietary Consumption of Garlic on Delaying Cataract Induced by Sodium
Selenite in Rats
1Fatma H. Abd El-Razek, 1Nora M. El-Sheikh, 1Tahany E. Kholeif, 2Mohamed S. Al-Balkini,
2Anhar M. Gomaa and 1Hasnaa H. Hassan
1Biochemistry and Nutrition Department, Women's College-Ain-Shams University;
2Ophthalmic and Biochemistry Department, Research Institute of Ophthalmology,
Abstract: Commercial garlic is widely used for certain therapeutic purposes, including cardiovascular
disorders, anti-microbial, anti-cancer, and treatment of hyperglycemia. Garlic posses a strong
antioxidant protective effect by its ability to scavenge free radicals. Several studies reported the
correction of serum lipid profile in response to consumption of garlic powder, thus assumed to have
a protective effect against atherosclerosis. The present study aims to investigate the role of dietary
consumption of garlic on delaying or protecting from cataract formation. Materials and Methods:
Twenty seven Wister rat pups were divided into four groups. Group one (n=6) received basal diet and
served as control. The rats in group 2 (n=8) were injected subcutaneously with sodium selenite
(30µmol/kg body weight) to induce cataract and fed on basal diet. Group 3 (n=6) were given 5%
garlic powder added to their basal diet. Group 4 selenite-induced cataract (n=7) also administered 5%
garlic powder in their basal diet. Development of cataract was assessed one week later, and its density
was graded by slit lamp biomicroscopy. Total phenolic compounds of garlic were determined. After
the end of experiment (two months), all rats were fasted overnight. Blood samples were collected from
the eye vein and then the crystalline lenses were excised. All planned samples were prepared as will
be described. The levels of serum lipid profiles were determined. The antioxidants and oxidative stress
parameters such as superoxide dismutase, catalase, reduced glutathione, total antioxidant capacity,
malondialdehyde and nitric oxide were assessed. Fas ligand (FAS-L) as apoptotic marker was also
assessed in the blood and lens. The crystalline lens protein patterns on sodium dodocyle sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE) were identified and analyzed by computerized
program. Results: All control rat lenses were clear. Development of cataract was reduced by 85.7%
in the group supplemented with 5% garlic. Selenite induced cataract group developed bilateral
complete opacification. The consumption of 5% garlic in cataractous group led to significant decrease
in serum total lipids (15.5%), total cholesterol (TC) 10.3%, triacylglycerol (TAG) 26.7%, and low
density lipoprotein cholesterol (LDL-C) 19.5%. There was a significant increase in the level of serum
high density lipoprotein cholesterol (HDL-C) 18.0% when compared to selenite-induced cataract
group. Also significant decrease in the activities of catalase, superoxide dismutase, reduced glutathione
and total antioxidants. A significant increase in the levels of malondialdehyde, nitric oxide and Fas-L
were noticed in cataractous group when compared to control group. The levels of all previous
parameters were improved after treatment with 5% garlic. Conclusion: Our findings indicate that garlic
inhibits selenite-induced cataract formation by inhibiting lipid peroxidation, oxidative stress and act
as anti-apoptotic agent that can delay progress of cataract formation.
Key words: garlic-selenite-induced cataract- phenolic compounds- oxidative stress-lipid profile-Fas-L-
nitric oxide- delay cataract.
INTRODUCTION
Garlic was found in Egyptian pyramids and ancient Greek temple. Medical applications of garlic were
mentioned in India, Egypt and Rome (Rivlin et al., 2006). Interest has increased considerably in finding
naturally occurring antioxidants for use in foods, cosmetics or medicinal materials to replace synthetic
antioxidants, which are being restricted due to their carcinogenicity (Sasaki et al., 2002). Garlic has strong
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1345
antioxidant properties and it has been suggested that garlic can prevent different diseases (Jalal et al., 2007
and Jung et al., 2008).
With regard to cataract, the selenite model was selected because of the rapid, effective and reproducible
cataract formation. Although the rate of opacification in the selenite model is much more rapid than in human
cataract, it has many general similarities to human cataract (Shearer et al., 1983 and 1997). Selenite induces
bilateral nuclear cataract within 4 to 6 days when administrated to suckling rat pups before completion of the
critical maturation period of the lens.
In cataractous state, an enormous production of reactive oxygen species takes place leading to characteristic
membrane permeability and changes including leakage of structural proteins which is implicated in the opacity
of the lens. The opacification of the crystalline lens occurs by aggregation of cytoplasmic lens proteins due
to modifications in the intermolecular interactions that include oxidative stress (Shearer et al., 1997). The role
of garlic in preventing age-related diseases has been investigated extensively over the last 10–15 years. Alireza
et al. (2009) evaluated the inhibitory impacts of the aqueous extract of garlic on the formation of cataract
induced by sodium selenite and suggested that, such herbal remedy may be considered for treatment of cataract.
This study aims to investigate the way and the role of garlic dietary consumption that can decrease or
delay selenite-induced cataract formation rate in pup rats.
MATERIALS AND METHODS
Diet Preparations:
The experiment was done in the animal house of Research Institute of Ophthalmology. A commercial diet
was used as basal diet. This diet consists mainly of 21% protein, 6 % fat, 3% fiber, and 6% of vitamins and
minerals mixture, methionine and choline chloride. The other type of diet was prepared by addition of 5%
Garlic powder to the diet as mentioned by Kweon et al. (2003). Dried powdered garlic was purchased from
Spicy Trade Company, Helwan, Cairo.
Experimental Design:
Twenty Seven Wister rat pups with an average body weight of 25±4 g were obtained from the animal
house of Research Institute of Ophthalmology. Four rat mothers having rat pups aging 10±1 days were included
in this study. Each rat mother and their pups were housed in one cage and served as groups from one to four.
The Experiment Included Four Groups:
Group (a) control fed on basal diet (6 pups). Group (b) selenite-induced cataract fed on basal diet (8
pups). Group (c) Positive control fed on basal diet containing 5% garlic (6 pups). Group (d) selenite-induced
cataract fed on basal diet containing 5 % garlic (7 pups).
The rat pups in the experimental groups (b&d) received a single subcutaneous injection of sodium selenite
(30µmol/kg) according to the method described by Orhan et al. (1999), while control group was injected with
normal saline (0.3 ml). Water and diets were available ad libitum for all rat pups. The progression of cataract
was under observation till the end of the experiment.
Slit Lamp Biomicroscopic Examination and Lenticular Opacification:
At the final examination, the pupils were dilated with tropicamide (0.5%) and phenylephrine hydrochloride
(2.5%). To assess the onset and maturation states of cataract, slit lamp biomicroscopic examination was carried
out at regular intervals and the stages were designated as described by Suryanarayana et al. (2005). Briefly,
lenses were examined on alternate days and opacities observed were graded into four stages: clear, stage 0;
no vacuoles present or clear lens, stage 1: vacuoles of less than one third of the lens radius, stage 2: vacuoles
located at the periphery of the lens occupying an area between one third and two thirds of the radius from the
periphery, stage 3: vacuoles extending up to two thirds of the radius from the periphery (nuclear opacity may
be seen), stage 4: vacuoles cover the entire lens, which appears white to the naked eye. The incidence of
cataract appearance was expressed as the percentage of total lenses in each group.
Blood Samples:
At the end of experiment (two months), the rats were fasted overnight, anaesthetized and blood samples
were withdrawn from the eye vein and divided into two portions:
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1346
The first portion was collected into sterile dry tube to separate serum by centrifugation at 3500 rpm and
kept in deep freezer under –30oC to be used to determine lipid profile, total antioxidant capacity,
malondialdehyde, nitric oxide, FAS-L and electrophoreses of lens protein.
The second portion (1ml) was taken in vacutest sterile tube with EDTA interior for the determination of
the superoxide dismutase activity and reduced glutathione. Plasma was used for determination of catalase
activity.
The eyes were enucleated, the lenses were excised, carefully decapsulated and washed in 0.15 M isotonic
sodium chloride solution. Lens homogenate was prepared according to each method for different determinations.
Biochemical Analyses:
Serum total lipids (TL) was determined according to the method described by Zollner and Kirsch (1962),
triacylglycerols (TAG) was analyzed according to the method described by Fossati and Prencipe (1982), serum
total cholesterol (TC) was measured according to the method of Allain et al. (1974), high density lipoprotein-
cholesterol (HDL-C) was measured by using method of Lopes-Virella et al. (1977), and low density
lipoprotein-cholesterol (LDL-C) was estimated by Tietz (1999).
Catalase activity (CAT) was determined according to the method described by Aebi, (1984); superoxide
dismutase activity (SOD) was assessed by using the method described by Marklund and Marklund (1974); total
antioxidants capacity (TAO) was measured according to the method of Koracevic et al. (2001); reduced
glutathione (GSH) was assessed according to the method of Beutler et al. (1963); malondialdehyde (MDA)
was determined according to the method described by Draper and Hadley (1990); and nitric oxide (NO) was
assessed according to the method of Moshage et al. (1995). APO-1 Fas was assessed in serum and lens by
a competitive enzyme linked immuno-sorbant assay (ELISA) Kit, using human (APO-1 Fas) as standard to
assess apoptosis according to the manufacturer’s instructions (Biosource International, INC., Camarillo,
California, U.S.A.) (Tanaka et al., 1996).
Analysis of Eye Crystalline Lens Proteins:
The protein patterns of the soluble fraction of the lens homogenate were analyzed by using sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970). A 12% (w/v)
polyacrylamide separating gel and 4% (w/v) polyacrylamide staking gel were used. Coomassie Brilliant Blue
was used to detect the lens protein bands. The gels were scanned, photographed and analyzed using Bioimage
software. Total protein was estimated according to the method described by Lowry et al. (1951).
Identification of Phenolic Compounds by HPLC:
The phenolic compounds present in garlic sample were identified according to the method described by
Duke et al. (2003). A known weight of air dried plant sample was used. Identification of individual phenolic
compounds of the garlic was performed on JASCO HPLC, using hypersil C18 reversed-phase column (250 x
4.6 mm) with 5 particle size.
Statistical Analysis:
All statistical calculations were carried out with the statistical package for social sciences (SPSS) software
program (version 10.0 for Windows). The values are expressed as the mean ± SE. The data were statistically
analyzed using analysis of variance (ANOVA) and significant difference of the means was determined using
Duncan’s multiple range tests at the level of P< 0.05.
Results:
Slit Lamp Examination and Degree of Opacification:
Table 1: Grading of Cataract on the Basis of Slit-Lamp Examination.
Groups Number of rats Stages of cataract % of cataract
-----------------------------------------------------------------------------------
Stage0 Stage1 Stage2 Stage3 Stage4
Normal 6 6 - - - - 0%
Cataract 8 - - - - 8 100%
Normal fed on garlic 6 6 - - - - 0%
Cataract fed on garlic 7 6 1 - - - 14.3%
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1347
Table (1) shows cataract grade distribution in experimental groups. All control rat lenses were clear - stage
zero (Figures 1-1 and 1-2). In selenite cataract group eight of eight rats 100% developed bilateral stage 4
cataract (Figure 1-3) whereas one rat only developed a bilateral 14.3% of lenses- stage 1 in selenite cataract
group fed on 5% garlic (Figure 1-4).
Fig. 1: Cataract formation observed in one sample from each group.
1: control group; 2: selenite cataract group; 3: control fed on 5% garlic; 4: selenite cataract fed on 5% garlic
Phenolic Compounds in Garlic:
Table 2: Phenolic Compounds in Garlic.
Phenolic Compounds mg/100g garlic Phenolic Compounds mg/100g garlic
Pyrogallic acid 780 Pinostrobin 510
Salicylic acid 2030 Daidzin 90
Protocatechuic acid 1440 Genistein 40
P-cumuric acid 140 Catechines 760
Eugenol 1660 Genistin 40
Quercetin 150 Myricetin 120
Pinocembrin 60 Rutin 380
3,5 dihydroxy isoflavone 390 Luteolin 200
Table (2) shows that, garlic contains high amounts of salicylic acid, eugenol, protocatechuic acid,
pyrogallic acid, catechines, pinostrobin, 3,5 dihydroxy isoflavone, rutin, luteolin, and P-cumuric acid. Garlic
also contains appreciable amount of the flavonids myricetin and quercetin. Also Daidzin, Genistein and Genistin
were found in garlic in a low concentrations.
Lipid Profile:
Table (3) shows the effect of supplementation with 5% garlic on serum total lipids, triacylglycerols, total
cholesterol, high and low density lipoprotein-cholesterol in the control and experimental groups. There were
significantly high levels of all lipid parameters except HDL-C in cataractous group as compared to the control
group (P<0.001-0.0005). It was interesting to note that the supplementation with 5% garlic to normal rats
showed a significant decrease in the levels of all previous lipid parameters. The percentage decreases were
19.9%, 9.1% and 24.4% concerning total lipids, total cholesterol and triacylglycerols, respectively. Also the
cataractous rats fed on 5% garlic (group d) showed significant decrease in the levels of all lipid parameters
except the level of HDL-C compared to cataractous group. The level of HDL-C in cataractous group showed
improvement by 18.0% after feeding 5% garlic (table 3).
Antioxidant Parameters:
The data in table (4) reported that, the mean values and ±SE of catalase was (283.8±8.45U/L), superoxide
dismutase activity (111.0±2.16 U/ml), reduced glutathione (65.2±0.82mg/dl) and total antioxidants capacity
(1.13±0.07mmol/L) in control group. A significant decrease in the levels of catalase (156.2±9.15, P<0.0007),
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1348
superoxide dismutase (73.2±3.90, P<0.0002) and total antioxidants (0.49±0.03, P<0.0001), and reduced
glutathione (44.9±0.56, P<0.0001) was noticed in cataractous group. Meanwhile, the percent of change were
-44.9%, -34.0%, -56.6% and -31.1% respectively when compared to control group.
Table 3: Means±S.E., P Values and % of Change for Serum Total Lipids (TL), Total Cholesterol (TC), Triacylglycerols (TAG), High
Density Lipoprotein-Cholesterol (HDL-C), and Low Density Lipoprotein-Cholesterol (LDL-C) in Control and Experimental
Groups.
Groups Normal Cataract Normal fed Cataract fed P-values (significant
Parameters (a) (b) on garlic (c) on garlic (d) differences between
groups)
TL mean±S.E 372.1±21.04 478.3±15.99 298.1±16.96 404.4±12.27 a,b (<0.0005);
mg/dl a,c(<0.006);
%Change 28.5%* -19.9%* 8.7%*
-15.5%# a,d (N.S);
d,b(<0.002).
TC mean±S.E 90.1±2.03 116.1±3.59 81.9±1.96 104.1±3.22 a,b (<0.0002);
mg/dl a,c(<0.049);
%Change 28.8* -9.1* 15.5%*
-10.3%# a,d (<0.003);
d,b (<0.006).
TAG mean±S.E 82.4±2.09 121.8±3.13 62.3±1.38 89.2±2.80 a,b (<0.0001);
mg/dl a,c(<0.0003);
%Change 47.8%* -24.4%* 8.3%*
-26.7%# a,d (N.S).
d,b (<0.0007).
HDL-C mean±S.E 43.3±1.34 39.9±1.49 51.4±1.42 47.1±2.14 a,b (N.S);
mg/dl a,c(<0.004);
%Change -7.8%* 18.7%* 8.7%*
18.0%# a,d (N.S);
d,b(<0.004).
LDL-C mean±S.E 31.3±2.12 49.8±2.62 19.5±1.73 40.1±2.21 a,b(<0.0001);
mg/dl a,c(<0.0003);
%Change 59.1%* -37.7%* 28.1%* a,d (0.004);
-19.5%# d,b(<0.001).
The asterisk (*) denotes that data and the percentage change compared to control group. The asterisk (#) denotes the percentage change
is compared to selenite-induced cataract group. The p values significantly different at P<0.05.
The results of the control fed on garlic showed a significant increase in the levels of catalase, superoxide
dismutase, reduced glutathione and total antioxidants capacity, the mean and ± S.E. values were 325.0±18.3
U/L; 127.4±4.22 U/ml; 76.2±0.61mg/dl and 1.42±0.02mmol/L, respectively.
After selenite injection of rats fed on garlic, the levels of catalase, superoxide dismutase, reduced
glutathione and total antioxidants capacity showed significant improvement with a percentage value of 44.8%,
19.9%, 24.9% and 102.9%, respectively as compared to cataract group (table 4).
Oxidative Stress and Apoptotic Marker:
The data in table (5) shows that, in cataract group, the levels of malondialdehyde, nitric oxide, and Fas-L
was significantly high. The mean ±SE of malondialdehyde (4.38±0.13); nitric oxide (4.16±0.07); and Fas-L
(467.5±5.91) were noticed in cataractous group. The percentage changes were 76.6%, 34.2% and 34.2%,
respectively as compared to control group. The percentage reductions observed in rats fed on garlic were -
18.9%, -27.4% and -21.0%, respectively compared to control group (table 5). Cataract group fed on garlic
showed a significant reduction in the levels of malondialdehyde, nitric oxide, and Fas-L. The mean±SE of
malondialdehyde (3.05±0.06, P<0.0001), nitric oxide (3.24±0.06,p<0.0004); and Fas-L (375.7±5.71, p<0.0002)
were reported. The percentages reductions were -30.4%, -22.1% and -19.6%, respectively.
Biochemical Assessment of Lens Crystalline:
Results in table (6) show that, lenses of cataract group demonstrated a marked reduction in levels of lens
total protein (represented as % of change), catalase activity, superoxide dismutase activity, total antioxidants
and reduced glutathione as compared to lenses of control group. Cataractous lenses (represented as % of
change) also showed the marked increase in levels of malondialdehyde, nitric oxide and Fas-L (represented
as % of change) compared to lenses of control group.
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1349
Table 4: Means ±S.E., P Values and % of Change for Catalase (CAT), Superoxide Dismutase Activities (SOD), Total Antioxidants
(TAO) and Reduced Glutathione (GSH) in Control and Experimental Groups.
Groups Normal Cataract Normal fed Cataract fed P-values (significant
Parameters (a) (b) on garlic (c) on garlic (d) differences between
groups)
CAT mean±S.E. 283.8±8.45 156.2±9.15 325.0±18.3 226.2±9.38 a,b(<0.0007;
a,c(<0.011);
U/L a,d (0.0003);
%Change -44.9%* 14.5%* -20.3%* d,b(<0.0006)
44.8%#
SOD mean±S.E. 111.0±2.16 73.2±3.90 127.4±4.22 87.8±2.45 a,b(<0.0002; a,c(<0.0003;
U/ml a,d(<0.0005;
%Change -34.0%* 14.7%* -20.9%* d,b (<0.003).
19.9%#
GSH mean±S.E 65.2±0.82 44.9±0.56 76.2±0.61 56.1±0.62 a,b(<0.0009; a,c(<0.0001;
mg/dl a,d(<0.0002)
%Change -31.1%* 16.3%* -14.0%* d,b(<0.0001)
24.9%#
TAO mean±S.E. 1.13±0.07 0.49±0.03 1.42±0.02 0.99±0.01 a,b(0.0001);
mmol/L a,c(<0.0008;
%Change - 56.6%* 25.7%* -12.4%*
a,d(<0.018); d,b(<0.0005)
102%#
Legends as shown in table (3).
Table 5: Means ±S.E., P Values and % of Change for Malondialdehyde (MDA), Nitric Oxide (NO) and Fas-L in Control and
Experimental Groups.
Groups Normal Cataract Normal fed Cataract fed P-values (significant
Parameters (a) (b) on garlic (c) on garlic (d) differences between
groups)
mean±S.E. 2.48±0.14 4.38±0.13 2.01±0.11 3.05±0.06 a,b(<0.0001;
a,c(<0.015);
MDA a,d (0.002);
nmol/ml %Change 76.6% * -18.9% * 20.9%* d,b(<0.0001).
-30.4%# a,b(<0.00005);
mean±S.E. 3.10±0.05 4.16±0.07 2.25±0.08 3.24±0.06 a,c(<0.0003);
NO
μmol/L %Change 34.2%* -27.4%* 4.5%%* a,d(<0.026);
-22.1%# d,b (<0.0004).
mean±S.E. 348.3±6.01 467.5±5.91 275.0±5.63 375.7±5.71 a,b(<0.00002);
a,c(<0.0001);
Fas-L
Pg/ml %Change 34.2%* -21.0%* 7.9%* a,d (0.002);
-19.6%# d,b(<0.0002).
Legends as shown in table (3).
Feeding 5% garlic to control rat pups caused a small increase in the levels of total lens proteins, catalase,
superoxide dismutase activities, total antioxidants capacity and reduced glutathione as compared to lenses of
control group; while it produced reduction in levels of malondialdehyde, nitric oxide and Fas-L as compared
to lenses of normal control group.
In case of cataract group fed on garlic the data showed a low reduction in the levels total lens protein,
catalase activity, superoxide dismutase activity, total antioxidants and reduced glutathione as compared to
control group; while it produced increase in levels of malondialdehyde, nitric oxide and Fas-L when compared
to lenses of control group.
Effect of Garlic on Lens Protein Profile:
In cataractous lenses, aggregations of proteins are related to lens membrane leakage. The selenite cataract
group rats showed a significant decrease in total protein in comparison to the controls (Table 6). There was
a remarkable increase of total protein contents in garlic-administered rats (control and selenite induced cataract
fed on garlic). The SDS-electrophoresis of the soluble protein fraction showed an aggregated band at 21.2 kDa
in relation to the controls and with a clear disappearance of this band in control group and selenite groups fed
on garlic (Fig. 2).
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1350
Table 6: Mean and Percentage of Change for Total Proteins (TP), Catalase Activity (CAT), Superoxide Dismutase Activity (SOD), Total
Antioxidants (TAO), Reduced Glutathione (GSH), Malondialdehyde (MDA), Nitric Oxide (NO), and Fas-L in the Soluble
Fraction of the Eye Lens Homogenate of Control and Experimental Groups.
Parameters TP CAT SOD TAO GSH MDA NO Fas-L
Groups mg/g lens U/ mg U/ mg mmol/ mg μg/ mg nmol/ mg μmol/ mg Pg/ mg
Lens Lens lens Lens lens lens protein lens
protein protein protein protein protein Fas-L protein
Normal Mean 0.67 1.2 1.26 9.4 108.7 3.3 0.65 4.07
Cataract Mean 0.47 0.7 0.66 7.1 65.8 5.8 1.24 6.9
% -30.5 -41.7 -47.6 -24.5 -39.5 74.2 90.7 69.5
Change
Normal Mean 0.71 1.4 1.36 9.6 120.6 2 0.35 3.17
with garlic % 5.2 16.7 7.9 2.1 10.9 -39.9 -46.15 -22.1
Change
Cataract Mean 0.58 1.05 1.12 8.8 94.9 3.4 0.67 5.17
with garlic % -14.1 -12.5 -11.1 -6.4 -12.7 2.1 3.1 27
Change
*The mean of duplicate sample.
Fig. 2: The effect of 5% garlic powder on protein cross-linking and aggregation of the soluble fraction of the
lens. The arrow indicates the cross-linked proteins.1) normal lens, 2) normal fed on garlic, 3) cataract
fed on garlic and 4) Cataractous lens.
Discussion:
Cataracts, the opacification of the eye lens, are the most common cause of blindness, accounting for almost
half of all cases worldwide (Bethesda, 1998) At present, the treatment for cataracts requires removal of the
natural lens that has developed opacification, through surgery, and replacing it with a synthetic lens to restore
the vision. Treatment is relatively expensive and there is a significant rate of postsurgical complications (Hirsch
and Schwartz, 1983) Therefore, alternative treatments must be used. To date, as a part of better strategic
management of cataract, metabolic intervention through natural dietary ingredients is gaining importance in
recent times (Vibin et al., 2010; Pourkabir et al., 2010; Joshua et al., 2011).
Garlic is the oldest of all cultivated plants and is widely used because of its high pharmacological
significance. Thus, the present study aimed to have an insight on the ameliorative action of garlic against
selenite-induced cataract in rats.
Oxidative stress is the result of an imbalance of antioxidants and pro-oxidants. Lens opacity, due to
cataract formation, is directly attributed to oxidative processes that occur within the lens. Oxidation, which can
be caused by an over abundance of oxidative stress generators, such as molecular oxygen, hydrogen peroxide,
and free radicals, produces major insult upon the crystalline lens, which can lead to the loss of glutathione,
lipid peroxidation, and decrease in antioxidant enzymes activity (Bhuyan and Bhuyan, 1984; Meister, 1991;
and Mitton et al., 1993).
We have evaluated the effects of garlic in the inhibition of cataracts formation induced by sodium selenite
in rat pups. Results from ophthalmic examination indicate that 5% garlic powder is able to prevent, or at least
significantly reduce the opacification of the lens crystalline within an experimental cataract model. This premise
is very evident from our study, in that 85.7% of the rats supplemented with 5% garlic did not develop any
opacification of the lens, whereas 14.3% of lenses developed cataract (table 1, figure 1 (4). The one hundred
percentage of the rat lenses treated with sodium selenite developed lenticular opacification (table 1, figure 1(2).
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1351
In the present study the phenolic compounds present in garlic sample were identified by HPLC. Sharma
et al. (1998) and Rhone and Basu (2008) supported the safety of higher intakes of the phytochemicals
catechines and its association with reducing risks of cataracts. Recently Cotlier (2011), reported that salicylic
acid lowers blood tryptophan that may delay or prevent the formation of senile cataracts. The observed findings
in this study could be well because of the ameliorative action of the bioactive compound salicylic acid,
eugenol, protocatechuic acid, pyrogallic acid and catechines as shown in table (2) and suggest that garlic is
efficient potential antioxidant against the changes imposed by cataract formation. Our results are in agreement
with previous findings (Vinson et al., 1998; Jackson et al., 2002; and Eidi et al., 2006).
A previous report by Bhuyan and Bhuyan (1984) and Babizhayev (1996) found a close relationship among
cataract and hyperlipidemia. Also lipid peroxidation has been associated with the cataracts formation. Intake
of garlic powder reduced total cholesterol, LDL-C and triacylglycerol and increased HDL-C (Liu and Yeh
2001; and Kojuri et al., 2007). Elkayam et al. (2003) suggested that garlic prevents hypercholesterolemia.
Similarly Pourkabir et al. (2010) showed that supplementation with 5% garlic powder for 8 weeks had effect
on the lipid profile in the cataractous rats. Our study supported these findings, the results indicate a reduction
of about 15.5%, 10.3%, 26.7%, and 19.5% in serum TL, TC, TAG, and LDL-C, respectively and an increase
by 18.0% of HDL-C level in response to 5% garlic powder was noticed. These results indicate that routine
consumption of garlic in the diet has a beneficial effect in maintaining the serum lipids at low or normal levels
and prevent lipid peroxidation and in turn cataract formation.
The glutathione redox system is a major component of overall antioxidant defenses in the cells. It is very
efficient free-radical scavenger and protects cells from the toxic effects of reactive oxygen compounds (Lang
et al., 2000). The selenite induced cataracts in pups have lower concentrations of glutathione in whole blood
and lens as reported in this study. The data demonstrated that cataractous group have been linked directly to
impaired glutathione status, and garlic powder has been shown to increase glutathione in the blood and lens.
Garlic being a strong antioxidant can participate in prevention cataract by its ability to scavenge free radicals,
and enhance scavenging systems in the lens, including the activity of each of superoxide dismutase, catalase
and reduced glutathione.
In addition, SOD and catalase were found to act as defense enzymes to the oxidative stress in cataract.
Significant increase was observed in the activity of previous enzymes (tables 4 and 6) accordingly profound
protection against peroxidation damage in the lens upon treatment with 5% garlic powder.
This effect was associated with higher GSH level as well as lower levels of MDA and nitric oxide in
response to garlic in normal and cataract groups (tables 4, 5 and 6) as compared to the cataract group. It could
be postulated that garlic powder act as a potent source of antioxidant which provide an additional support to
the elevation of SOD, catalase activities, and GSH level and decrease in MDA and nitric oxide levels.
Selenite-induced oxidative stress (30µmol/kg body weight) causes nuclear opacity through the calpain
proteolysis of lens proteins. It is a strong sulfhydryl oxidant. This study suggested that the role of garlic in
delaying cataract formation may be due to the active components which have potent antioxidant properties for
maintaining sulfhydryl groups (-SH) of crystalline lens proteins in their reduced form preventing disulfide cross-
linkage and prevent aggregation of lens protein. On the other hand, Orhan et al. (1999) reported that GSH
protects the structural proteins and enzymes from sulfhydryl cross-linking that can disrupt their function.
Considering the above facts, results from this study indicate that treatment with garlic decreases the oxidative
damage and can prevent the formation of cataracts by maintaining GSH levels.
Apoptosis (Programmed cell death) is involved in a whole array of normal physiologic processes, including
immune defense, tissue homeostasis, and development, and any tilt of the balance between life and death within
an organism can lead to disease. Thus, the loss of essential cells of postmitotic tissues due to enhanced cell
death may play an important role in a number of functional deficiencies and degenerative diseases such as
cataract (Gosslau and Chen 2004). Garlic also inhibits the proliferation of human cells and induces apoptosis
by increasing intracellular calcium concentrations (Sundaram and Milner 1996). Cho et al. (2006) found that
Allicin a major component of garlic inhibits apoptosis of macrophages in a depleted nutritional state. Apoptosis
as indicated by the level of serum Fas was assessed in the present study and the data shows a decrease in the
levels of FAS-L in induced selenite cataract rat after feeding with garlic (5%). Garlic exerts anti-apoptotic
action in different ways, due to the variety of compounds present in it such as water and lipid-soluble
organosulfur compounds, phenolic compounds, saponins and selenium.
Results in the present study show that the consumption of 5% garlic could protect the animal models
against selenite induced cataract. It is proved that garlic contain compounds that are effective antioxidant
against oxidative insult. In addition the understanding of the anti-apoptotic, hypolipidemic, and antioxidants
action of its active compound could open doors for further investigations in the management of cataract and
quality of vision.
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1352
In conclusion, the present biochemical and morphological findings prove that: garlic consumption appeared
effective to prevent Selenite induced cataract, perhaps through its potent free radical scavenging anti-apoptotic
and antioxidant properties.
REFERENCES
Aebi, H., 1984. Catalase in vitro. Methods in Enzymol., 105: 121-126.
Alireza, J., G. Amir, A. Sara, R. Nadereh, M. Mehran, R. Mandana, O. Yadollah, 2009. Prevention of
selenite-induced cataractogenesis in Wistar albino rats by aqueous extract of garlic. J. Ocular Pharmacology
and Therapeutics, 25(5): 395-400.
Allian, C.C., L.S. Poon, G.S.C. Chan, W. Richmond and P.C. Fu, 1974. Enzymatic determination of total
serum cholesterol. J. Clin. Chem., 20: 470-475.
Babizhayev, M.A., 1996. Failure to withstand oxidative stress induced by phospholipid hydroperoxides as
a possible cause of the lens opacities in systemic diseases and ageing. Biochim Biophys Acta, 1315: 87-99.
Bethesda, M.D., 1998. Vision research: a national plan 1999–2003: report of the National Advisory
Council. National Eye Institute, 59: 15-18.
Beutler, E., O. Doaron, D.M. Kelly, 1963. Improved method of the determination of blood glutathione.
J. Lab. Clin. Med., 61: 882-888.
Bhuyan, K.C. and D.K. Bhuyan, 1984. Molecular mechanisms of cataractogenesis. III. Toxic metabolites
of oxygen as initiators of lipid peroxidation and cataract. Curr. Eye Res., 3: 67-81.
Cho, S.J., D.K. Rhee, S. Pyo, 2006. Allicin a major component of garlic, inhibits apoptosis of macrophages
in a depleted nutritional state. Nutrition, 22: 1177-84.
Cotlier, E., 2011. International ophthalmology in rheumatoid arthritis and cataract. Surgery, 31: 127-129.
Draper, H.H. and M. Hadley, 1990. Malondialdehyde determination as index of lipid peroxidation. Methods
in enzymol., 186: 421-431.
Duke, S.O., A.M. Rimado, P.F. Pace, K.N. Reddy, K.J. Semeda, 2003. Isoflavone, glyphosate and
aminomethylphosphonic acid levels in seeds of glyphosate treated, glyphosate-resistant soybean. J. Agric Food
chem., 51: 350-354.
Eidi, A., M. Eidi, E. Esmaeili, 2006. Antidiabetic effect of garlic (Allium sativum L.) in normal and
streptozotocin-induced diabetic rats. Phyto Med., 13: 624-9.
Elkayam, A., D. Mirelman, E. Peleg, M. Wilchek, T. Miron, A. Rabinkov, M. Oron-Herman, T. Rosenthal,
2003. The effect of allicine on weight in fructose- induced hyperinsulinemic, hyprlipidemic, hypertensive rats.
AJH., 16: 1053-1056.
Fossati, P. and L. Prencipe, 1982. Serum triglycerides determination colorimetrically with an enzyme that
produces hydrogen peroxide. Clin Chem., 28: 2077-2080.
Gosslau, A. and Chen KYu., 2004. Apoptosis, cancer, and overexpression of proteins. nutraceuticals,
apoptosis, and disease prevention. Nutrition, 20: 95-102.
Hirsch, R.P. and B. Schwartz, 1983. Increased mortality among elderly patients undergoing cataract
extraction. Arch. Ophthalmol., 101: 1034-1037.
Jackson, R., B. McNeil, C. Taylor, 2002. Effect of aged garlic extract on caspase-3 activity in vitro. Nutr
Neurosci., 5: 287-290.
Jalal, R., M.S. Bagheri,, A. Moghimi, M.B. Rasuli, 2007. Hypoglycemic effect of aqueous shallot and
garlic extracts in rats with fructose-induced insulin resistance. J. Clin. Biochem. Nutr., 41: 218-223.
Joshua, W., A. Carey, Y. Eylem, B. Pinarci, P. Suman, K. Humeyra, E. Nuran, 2011. Pharmacological
effects of garlic (Allium sativum L.). Annual review of In vivo inhibition of l buthionine-(S,R)-sulfoximine-
induced cataracts by a novel antioxidant, N-acetylcysteine amide. Free Radical Biology & Medicine, 50: 722-
729.
Jung, S.H., K.D. Kang, R.J. Fawcett, Safa R., T.A. Kamalden, N.N. Osborne, 2008. The flavonoid baicalin
counteracts ischemic and oxidative insults to retinal cells and lipid peroxidation to brain membranes.
Neurochemistry International, 53: 325-337.
Kojuri, J., R. Vosoughi, M. Akrami, 2007. Effects of anethum graveolens and garlic on lipid profile in
hyperlipidemic patients. Lipids Health Dis., 6: 5-9.
Koracevic, D., G. Koracevic, V. Djordjevic, S. Andrejevic, V. Cosic, 2001. Method for the measurement
of antioxidant activity in human fluids. J. Clin. Pathol., 54: 356-361.
Kweon, S., K.A. Park, H. Choi, 2003. Chemopreventive effect of garlic powder diet in diethylnitrosamine-
induced rat hepatocarcinogenesis. Life Sci., 73: 2515-2526.
Aust. J. Basic & Appl. Sci., 5(8): 1344-1353, 2011
1353
Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4.
Nature, 2777: 680-685.
Lang, C.A., B.J. Mills, W. Mastropaolo, M.C. Liu, 2000. Blood glutathione decreases in chronic diseases.
J.Lab. Clin. Med., 135: 402-405.
Liu, L. and Y.Y. Yeh, 2001. Water-soluble organosulphur compounds of garlic inhibit fatty acid and
triglyceride synthesis in cultured rat hepatocytes. Lipids, 36: 395-400.
Lopes-Virella, M.F., P. Stone, S. Ellis, 1977. Cholesterol determination in high density lipoproteins
separated by three different methods. Clin Chem., 23: 882-886.
Lowry, O.H., N.J. Rosebrough, A.L. Farr, R.J. Randall, 1951. Protein measurement with the folin phenol
reagent. J. Biol. Chem., 193: 265-275.
Marklund, S. and G. Marklund, 1974. Involvement of the superoxide anion radical in the auto-oxidation
of pyrogallol and a convenient assay for superoxide dismutase. Eur.J. Biochem., 47: 469-474.
Meister, A., 1991. Glutathione deficiency produced by inhibition of its synthesis, and its reversal
applications in research and therapy. Pharmacol. Ther., 51: 155-194.
Mitton, K.P., P.A. Dean, T. Dzialoszynski, H. Xiong, S.E. Sanford, J.R. Trevithick, 1993. Modelling
cortical cataractogenesis. Early effects on lens ATP/ADP and glutathione in the streptozotocin rat model of the
diabetic cataract. Exp. Eye Res., 56: 187-198.
Moshage, H., B. Kok, J.R. Huizenga, P.L.M. Jansen, 1995. Nitrite and nitrate determinations in plasma:
a critical evaluation. Clin Chem., 41: 892-896.
Orhan, H., S. Marol, I.F. Hepsen,, 1999. Effects of some probable antioxidants on selenite-induced cataract
formation and oxidative stress-related parameters in rats. Toxicology, 139: 219-232.
Pourkabir, M., T. Shomali, F. Asadi, 2010. Alterations in serum lipid, lipoprotein and visceral abdominal
fat pad parameters of hypercholestrolemic guinea pigs in response to short term garlic consumption. African
J. Biotechnology, 9: 7930-7933.
Rhone, M. and A. Basu, 2008. Phytochemicals and age-related eye diseases. Nutr Rev., 66: 465-472.
Rivlin, R.S., M. Budoff, H. Amagase, 2006. Significance of garlic and its constituents in cancer and
cardiovascular disease. J. Nutr., 136: 713S-715S.
Sasaki, Y.F., S. Kawaguchi, A. Kamaya, M. Ohshita, K. Kabasawa, K. Iwama, 2002. The comet assay
with 8 mouse organs: results with 39 currently used food additives. Mutation Research/Genetic. Toxicology
and Environmental Mutagenesis, 519: 103-109.
Sharma, P., S. Kulshreshtha, A.L. Sharma, 1998. Anti-cataract activity of Ocimum sanctum on experimental
cataract. Indian Journal of Pharmacology, 30: 16-20.
Shearer, T.R., R.S. Anderson, J.L. Britton, E.A. Palmer, 1983. Early development of selenium- induced
cataract: slit lamp evaluation. Exp Eye Res., 36: 781-788.
Shearer, T.R., H. Ma, C. Fukiage, M. Azuma, 1997. Selenite nuclear cataract: review of the model. Mol
Vis., 3: 8-23.
Sundaram, S.G. and J.A. Milner, 1996. Diallyl disulfide induces apoptosis of human colon tumor cells.
Carcinogenesis, 17: 669-673.
Suryanarayana, P., M. Saraswat, T. Mrudula, 2005. Curcumin and turmeric delay streptozotocin-induced
diabetic cataract in rats. Investig Ophthalmol Vis Sci., 46: 2092-2099.
Tanaka, M., T. Suda, K. Haze, N. Nakamura, K. Sato, F. Kimura, K. Motoyoshi, M. Mizuki, S. Tagawa,
S. Ohga, K. Hatake, A.H. Drummond, S. Nagata, 1996. Fas ligand in human serum. Nat Med., 2: 317-322.
Tietz, N.W., 1999. Determination of LDL-C. Clinical guide to laboratory tests 3rd ed. Saunders CO.
Vibin, M., S.G. Siva Priya, B. Rooban, V. Sasikala, V. Sahasranamam, A. Abraham, 2010. Broccoli
regulates protein alterations and cataractogenesis in selenite models. Curr Eye Res., 35: 99-107.
Vinson, J.A., Y. Hao, X. Su, L. Zubik, 1998. Phenol antioxidant quantity and quality in foods: vegetables.
J. Agri. Food Chem., 46: 3630-3634.
Zollner, N. and K. Kirsch, 1962. Micro determination of lipids by the sulphophosphovanillin reaction. Exp.
Med., 135: 545-461.