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Food and Chemical Toxicology 49 (2011) 30983103

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Antioxidant and lipid peroxidation activities in rats fed with Aspergillus

carbonarius carotenoid
Anbarasu Kumar a, Akshatha Hosahalli Srikanta a, S.P. Muthukumar b, Umesh-Kumar Sukumaran a,
Vijayalakshmi Govindaswamy a,
Department of Food Microbiology, Central Food Technological Research Institute (Council of Scientic and Industrial Research), Mysore 570020, India
Biochemistry and Nutrition, Central Food Technological Research Institute (Council of Scientic and Industrial Research), Mysore 570020, India

a r t i c l e i n f o a b s t r a c t

Article history: Effect of feeding partially saturated canthaxanthin (PSC), puried from Aspergillus carbonarius mutant,
Received 21 April 2011 was studied using four groups of female albino rats (n = 6) for four weeks. While the control group
Accepted 1 September 2011 received basal diet ad libitum, , Groups I, II and III were fed with basal diet containing 50, 100 and
Available online 8 September 2011
250 ppm PSC, respectively. PSC feeding did not cause any signicant changes in food intake and there
was no gain in body weight either. PSC included in the diet signicantly decreased cholesterol in blood.
Keywords: There was 44.75% and 60.54% decrease in LDL-cholesterol in rats fed with 50 and 100 ppm carotenoid.
Hepatic ascorbic acid content increased by 44.59% in rats fed with 50 ppm PSC. Dietary PSC at
250 ppm lowered lipid peroxides by 19.49%. Activities of antioxidant enzymes, glutathione transferase
HDL/LDL and catalase were signicantly higher in serum and liver of PSC fed rats compared to the controls. The
Rats results suggested that PSC feeding can induce hypocholesterolmic and antioxidant properties in rats.
2011 Elsevier Ltd. All rights reserved.

1. Introduction is treated with drugs like lovastatin, pravastatin, uvastatin, sim-

vastatin, atorvastatin (Biccard et al., 2005), gembrozil (Frick
Oxygen, an indispensable part of aerobic life, under certain cir- et al., 1987) and nicotinic acid (Manninen et al., 1988).
cumstances can seriously affect human health due to the formation Nutraceuticals from natural products are gaining importance
of reactive oxygen species (ROS). The deleterious effects of both due to side effects of drugs used for treatment of hyperlipidemia
free and non-free radical species of oxygen are atherosclerosis, and oxidative stresses. Natural edible materials have also been
ischemic heart disease, aging, inammation, diabetes, immuno- identied for antihyperlipidemic and antioxidant effects (Hu
suppression, neurodegenerative diseases and cancer (Jadhav and et al., 2006). Supplementation of vegetable oils in diets enhance
Bhutani, 2002; Gulcin et al., 2002). Nevertheless, almost all organ- antioxidant activities in normal rats (Reena and Lokesh, 2011;
isms are protected from free radical attack by defense mechanisms Shireen et al., 2008). Evidences from animal studies have shown
such as preventive antioxidant system that reduce the rate of free curcumin of turmeric, capsaicin of red pepper (Manjunatha and
radical formation, and another system to produce chain-breaking Srinivasan, 2007), tocotrienols from cereals (Minhajuddin et al.,
antioxidants that scavenge and stabilize free radicals. When free 2005), carotenoids of carrot (Hu et al., 2006), dietary chitosan
radical production rate exceeds the capacity of the antioxidant de- (Hu et al., 2006), polyphenols of green tea (Lin et al., 1998) and sul-
fense mechanisms, substantial tissue injury results (Rahman and fur compounds of onion (Manjunatha and Srinivasan, 2007) to
Moon, 2007). Therefore, prevention and therapeutics of free radical have hypolipidemic and antioxidant properties.
mediated diseases are greatly dependant on the use of antioxidants Of the above, dietary carotenoids have a preference since they
with free radical scavenging activities. Antioxidants important in are colorants and preservative agents with typical avor. Though
the pharmaceuticals are butylated hydroxy toluene (BHT), butyl- there are more than 600 carotenoids in nature, only 10% of these
ated hydroxy anisole (BHA), ascorbic acid, a-tocopherol and their are involved in vitamin A conversion. Others possess several
analogues, etc. important functions that include antioxidant activities, promotion
In addition to oxidative disorders, improper lipid metabolism of cell differentiation, regulation of cell proliferation and intracel-
causes hyperlipidemia and related diseases. Peroxidation of lipid lular communication via gap junctions (Aust et al., 2003), regula-
is related to coronary heart disease. Hence, hypercholesterolemia tion of cellular activities thereby detoxifying enzymes and
enhancing immune function. The antioxidant properties of carote-
noids have also been shown using various in vivo and in vitro model
Corresponding author. Tel.: +91 821 2517539; fax: +91 821 2517233.
systems. Studies show that carotenoids have antioxidant property
E-mail address: (V. Govindaswamy).

0278-6915/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
A. Kumar et al. / Food and Chemical Toxicology 49 (2011) 30983103 3099

and can modify cholesterol absorption so also its oxidation (Nicolle Table 1
et al., 2003). In Deinococcus radiodurans, Fusarium aquaeductuum, The composition of basal diet.

Rhodotorula mucilaginosa and Phafa rhodozyma, carotenoids pro- Ingredient %

tected the organism against experimentally induced oxidative Corn starch 50.00
damage (Carbonneau et al., 1989; Theimer and Rau, 1970; Moore Casein 20.00
et al., 1989; Schroeder and Johnson, 1995; Kobayashi et al., Sucrose 13.00
1997). Nicolle et al. (2003) showed that carrot carotenoids in diet Ground nut oil 7.00
Cellulose 5.00
reduce plasma cholesterol and lipoprotein (LDL and VLDL) in Mineral mix 3.50
hypertensive rats fed with high cholesterol diet. Antioxidant activ- Vitamin mix 1.00
ity of astaxanthin is claimed to be 10 times stronger than that of L-Cystine 0.30
other carotenoids, zeaxanthin, lutein, canthaxanthin, and b-caro- Choline 0.25
tene (Miki, 1991).
The carotenoid, partially saturated canthaxanthin (PSC), of
Aspergillus carbonarius mutant, was found to induce apoptosis in
cancer cells that possessed retinoic acid receptors (Kumaresan 2.3. Experimental design
et al., 2008). Apparently due to its DPPH radical scavenging, lipid
peroxidation inhibition and metal chelating activities, the fungal Animals (n = 6), were randomly distributed into four groups and
biomass containing carotenoid was found safe when fed as a diet maintained on various experimental diets consisted of AIN-93G
to experimental rats at acute and sub acute levels (Sanjay et al., based diets with or without PSC ad libitum for four weeks. The
2007). This study revealed that PSC enhanced antioxidant proper- composition of basal diet is described in Table 1. Animals in group
ties and reduced cholesterol and inhibited lipid peroxidation when I that received basal diet served as control. Animals in group II,
fed to rats. The details are described in this paper. group III and group IV received basal diet containing 50, 100 and
250 ppm PSC, respectively. After the experiment, animals were
2. Materials and methods euthanized, blood was collected by heart puncture and serum
was separated by centrifugation at 4000 rpm for 20 min at 4 C. Li-
2.1. Extraction of carotenoid from A. carbonarius mutant ver was quickly excised, weighed, homogenized with phosphate
buffer and stored frozen till analyses. Serum and liver homoge-
The fungus A. carbonarius mutant accumulates PSC as a major nates were analyzed for total cholesterol (TC), triglycerides (TG),
carotenoid with traces of partially saturated astaxanthin when phospholipids, low density lipoprotein-cholesterol (LDL-C), high
grown in culture (Kumaresan et al., 2008). The carotenoids were density lipoprotein-cholesterol (HDL-C), very low density lipopro-
extracted from wet biomass with acetone:methanol (7:2) and con- tein-cholesterol (VLDL-C), lipid peroxides, antioxidant enzymes
centrated at 40 C using rotary evaporator (Buchi Rotovapor R-205, (glutathione peroxidase, glutathione transferase, glutathione
Switzerland). PSC was puried from the above by phase separation reductase, catalase) and antioxidant molecules (total thiols, ascor-
using hexane. The hexane layer collected and concentrated to dry- bic acid, glutathione and a-tocopherol).
ness, re-dissolved in methanol was quantied by HPLC by compar-
ing puried standard (Fig. 1) prepared by the method described by 2.4. Lipid prole
Kumaresan et al. (2008). The HPLC conditions were, acetoni-
trile:methanol:tetrahydrofuran (58:35:7) as mobile phase, C18 Total lipids were extracted according to Folch et al. (1957). Cho-
column, 2 ml ow rate and detection at 414 nm using photodiode lesterol, triglycerides and phospholipids were estimated in the li-
array detector. pid extracts of liver using the procedures described by Searcy
and Bergquist (1960), Fletcher (1968) and Charles and Stewart
2.2. Experimental animals (1980), respectively. Total serum cholesterol and triglycerides
were measured enzymatically using kits procured from Span Diag-
PSC taken in ground nut oil was incorporated in the basal diet nostics Ltd., India. The lipid prole of serum, including cholesterol,
(AIN 93-G) for feeding female Wistar rats (CFT strain) weighing triglycerides and phospholipids were determined using biochemi-
100 5 g and housed in individual stainless steel cages at room cal kits procured from Span diagnostics Ltd., India. The HDL frac-
temperature (25 2 C). Experiments were conducted in accor- tion of lipid extracts of serum and liver were estimated with a
dance with the guidelines prescribed by the Institutional Animal diagnostic D-HDL-C Reagent Kit as described by the manufacturer
Ethics Committee (IAEC No. 142/09). (Biosino Bio-Technology and Science Inc., China). The levels of LDL-
C and VLDL-C were calculated using Friedewald equation (Friede-
wald et al., 1972).

2.5. Lipid peroxides


Lipid peroxides in liver and serum were estimated by photo-

100 metric measurement of thiobarbituric acid complex extracted into

butanol (Ohkawa et al., 1979). Absorbance of the butanol extract

was measured at 532 nm and quantied using tetraethoxypropane
50 standard.

2.6. Antioxidant molecules
0 1 2 3 4 5
Total thiols in serum and liver homogenate were measured
spectrophotometrically using Ellmans reagent as described by
Fig. 1. HPLC elution of partially saturated canthaxanthin from A. carbonarius. Sedlock and Lindsay (1968). Ascorbic acid in liver homogenate
3100 A. Kumar et al. / Food and Chemical Toxicology 49 (2011) 30983103

was estimated spectrophotometrically by measuring 2,4-dini- Table 3

trophenylhydrazone derivative of dehydroascorbic acid (Omaye Effect of diet containing partially saturated canthaxanthin on liver lipid prole of
experimental rats1.
et al., 1973). a-Tocopherol in liver homogenate was assayed using
uorescence HPLC system with 295 nm emission wavelength and Animal Group Cholesteol Triglycerides Phospholipids
345 nm excitation wavelength, using 2% water in methanol as sol- (mg/g liver) (mg/g liver) (mg/g liver)

vent system delivered at a ow rate of 2.0 ml per min (Zaspel and Control 1.241 0.122 2.089 0.053bd 4.762 0.066
Csallany, 1983). For serum a-tocopherol, the HPLC method using Carotenoid (50 ppm) 1.221 0.051 2.072 0.126bc 4.756 0.047
Carotenoid (100 ppm) 1.115 0.099 2.273 0.109c 4.771 0.085
UVvisible detector monitored at 292 nm as described by Zaspel Carotenoid (250 ppm) 1.220 0.125 1.751 0.095a 4.883 0.084
and Csallany (1983) was used.
Values are means SD (n = 6). Values in a column not sharing a common
superscript are signicantly (P < 0.05) different from each other.
2.7. Antioxidant enzymes

Serum and liver glutathione reductase activities were assayed

by measuring NADPH oxidation at 340 nm due to glutathione oxi- The PSC concentration had no effect on serum VLDL-cholesterol
dation (Carlberg and Mannervik, 1985). Glutathione-S-transferase or HDL-cholesterol in rats. However, signicant (P < 0.05) decrease
activity was assayed by measuring CDNBGSH conjugate formed in serum LDL concentration was estimated in rats fed with 50 and
using 1-chloro-2,4-dinitrobenzene as substrate (Warholm et al., 100 ppm PSC (44.74% and 60.54%, respectively). Though not signif-
1985). Glutathione peroxidase activities in serum and liver homog- icant, there was a 37.86% decrease in serum LDL in rats fed with
enates were determined by following NADPH oxidation in a cou- 250 ppm PSC. Marginal increase in serum triglycerides (9.11%
pled reduction system consisting of hydrogen peroxide and and 25.08%) was recorded in rats fed with 50 and 100 ppm PSC,
oxidized glutathione (Flohe and Gunzler, 1984). Catalase activity respectively.
in serum and liver homogenate was assayed according to the Small but not signicant increase in phospholipid content was
method of Aebi (1984) wherein H2O2 decomposition was followed observed in carotenoid fed animals and the increase was 4.70%,
for a period of 3 min at 240 nm. 2.94% and 3.91% in the 50, 100 and 250 ppm fed animal groups,
2.8. Statistical analysis

Statistical analysis of the data was done using the SPSS statisti- 3.2. Liver lipid prole
cal package (7.5.1 version, SPSS Inc., 1996). One way ANOVA was
used for testing statistical signicance between groups of data, Hepatic total cholesterol, triglycerides and lipoprotein concen-
and individual pair difference was tested by means of Duncans trations of rats fed with PSC are described in Table 3. Hepatic cho-
multiple range test. All data presented are mean SD. A P value lesterol lowered as a result of carotenoid feeding in experimental
<0.05 was taken as statistically signicant. rats. The decrease was 1.61%, 10.15% and 1.69% in 50, 100 and
250 ppm carotenoid fed animals, respectively. However decreasing
triglycerides observed in 50 ppm fed animals become signicant
3. Results
(P < 0.05) in 250 ppm carotenoid fed rats. A profound increase
(P < 0.05) was observed in rats fed with 100 ppm PSC (8.81%).
Rats fed with 0.25% PSC in diet did not show any adverse effect
Though phospholipid concentration remained unaffected in 50
in sub-acute study. Based on this observation, feeding experiments
and 100 ppm dietary carotenoid fed groups 2.54% increase in its
were set up maintaining the maximum concentration of PSC at
concentration was recorded in animals fed with 250 ppm
250 ppm Rats fed with dietary PSC at 50, 100 and 250 ppm levels
were not affected in feed intake as like the animals maintained on
control diet. Similarly, no gain in body weight was recorded during
the 4 week carotenoid treatment. Serum SGOT, SGPT, alkaline 3.3. Antioxidant molecules in serum
phosphatase and creatinine kinase remained unchanged in rats
fed with PSC thus indicating its cardio protective function. Effect of carotenoid on serum antioxidant molecules and lipid
peroxides (Table 4) showed dose dependant increase in serum to-
3.1. Serum lipid prole tal thiols concentration. The extent of increase was 12.08%, 26.90%
and 41.12% in rats fed with 50, 100 and 250 ppm PSC, respectively.
Effect of A. carbonarius PSC feeding on serum lipid prole in Unlike the above, serum lipid peroxide content decreased to
experimental rats is presented in Table 2. PSC feeding at 50, 100 4.94% in animals fed with 50 ppm carotenoid. However increased
and 250 ppm decreased blood total cholesterol concentration feeding increased the blood lipid peroxide in 100 and 250 ppm die-
which was 19.42%, 16.05% and 16.42% lower in the respective tary carotenoid fed animals and the extent of increase was 5.12%
animal groups as compared to the control. and 0.37% in the respective diet groups.

Table 2
Effect of diet containing partially saturated canthaxanthin on serum lipid prole of experimental rats1.

Animal group Cholesterol (mg/dL) Triglycerides (mg/dL) Phospholipids (mg/dL)

Control 76.11 5.29a 29.37 5.59a 23.22 3.63 23.51 2.27 116.11 18.17 34.00 14.03
Carotenoid (50 ppm) 61.33 3.55b 16.23 3.45b 25.34 2.90 19.76 3.62 126.69 14.53 35.60 14.52
Carotenoid (100 ppm) 63.89 2.05b 11.59 7.24b 29.05 7.51 23.25 2.60 145.23 37.56 35.00 10.79
Carotenoid (250 ppm) 63.61 4.72b 18.25 8.37ab 23.37 4.54 21.99 3.24 116.85 22.72 35.33 12.75
Values are means SD (n = 6). Values in a column not sharing a common superscript are signicantly (P < 0.05) different from each other.
A. Kumar et al. / Food and Chemical Toxicology 49 (2011) 30983103 3101

Table 4 one reductase activity was affected (34.5756.40% according to

Effect of diet containing partially saturated canthaxanthin on serum antioxidant concentration of PSC fed). However the effect on serum glutathione
molecules and lipid peroxides of experimental rats1.
peroxidase activity was only marginal (15%). Unlike the above,
Animal group Total thiols a-Tocopherol Lipid peroxides the activity of serum glutathione transferase was signicantly
(mmol/dL) (lg/dL) (lmol/dL) inuenced by dietary carotenoid and higher quantity dietary feed-
Control 194.51 104.27 16.58 1.03 26.73 8.90 ing increased the enzyme activity by 2.98-fold. Increase in serum
Carotenoid 218.01 71.75 17.54 1.62 25.41 5.44 catalase activity was higher when fed with 50 ppm dietary carot-
(50 ppm)
Carotenoid 246.84 93.04 18.82 1.31 28.10 3.87
enoid (Table 6).
(100 ppm)
Carotenoid 274.49 79.36 17.55 0.79 26.83 1.65 3.6. Antioxidant enzymes in liver
(250 ppm)
Values are means SD (n = 6). Activities of the hepatic antioxidant enzymes, glutathione
reductase, glutathione peroxidase, glutathione transferase and cat-
alase when assayed it was found that (Table 7) the dietary caroten-
oid enhanced the activity of hepatic glutathione reductase (up to
3.4. Liver antioxidant molecules
22.6%), hepatic glutathione peroxidase (up to 90%), hepatic gluta-
thione transferase (up to 2.83-fold) and hepatic catalase (3.97-fold).
Dietary carotenoid fed to rats at 50 and 100 ppm increased total
thiols in liver to 10.81% and 3.47%. When fed at 250 ppm concen-
4. Discussion
tration, highest increase of 71.43% total thiols was recorded (Table
5). Increase in hepatic ascorbic acid content was signicant in
Tomato carotenoid, lycopene, when used as dietary supplement
experimental rats maintained on diet containing 50 ppm caroten-
at 60 mg/day signicantly decreased plasma LDL-cholesterol con-
oid. While ascorbic acid content increased by 44.59% in rats fed
centration without affecting HDL-cholesterol (Fuhrman et al.,
with 500 ppm carotenoid, 100 and 250 ppm diet feeding did not
1997). Likewise, Silaste et al. (2007) found that high daily dietary
result in corresponding increase. Unlike the above, hepatic
intake of tomato juice and ketchup for a period of 3 weeks reduced
a-tocopherol concentration in liver of rats fed with carotenoid in-
total cholesterol levels in healthy normocholesterolemic adults.
creased only marginally (Table 5).
They further reported that the average plasma total cholesterol
Dietary carotenoid signicantly lowered hepatic lipid peroxides
and LDL-cholesterol reduced by 5.9% and 12.9%, respectively,
only when fed at 250 ppm concentration (19.49%).
without any change in the concentrations of HDL-cholesterol and
triglycerides when fed with high tomato diet. The reduction in to-
3.5. Antioxidant enzymes in serum tal cholesterol observed in the rats fed with PSC was apparently
due to decreased serum LDL cholesterol as observed by Minhajud-
Partially saturated canthaxanthin feeding on serum antioxidant din et al. (2005). Like the lycopene the PSC may be suppressing
enzymes in experimental rats (Table 6) showed that the glutathi- cellular cholesterol synthesis in macrophages by directly inhibiting

Table 5
Effect of diet containing partially saturated canthaxanthin on liver antioxidant molecules and lipid peroxides of experimental rats1.

Animal group Total thiols (mmol/mg protein) Ascorbic acid (lg/mg protein) a-Tocopherol (ng/mg protein) Lipid peroxides (nmol/mg protein)
Control 0.259 0.047 3.999 0.496a 37.21 11.76 1.252 0.122a
Carotenoid (50 ppm) 0.287 0.031 5.782 0.309b 39.96 5.17 1.082 0.213ab
Carotenoid (100 ppm) 0.268 0.057 5.179 0.817ab 38.45 5.84 1.175 0.096ab
Carotenoid (250 ppm) 0.444 0.151 6.017 1.450ab 32.99 6.97 1.008 0.097b
Values are means SD (n = 6). Values in a column not sharing a common superscript are signicantly (P < 0.05) different from each other.

Table 6
Effect of diet containing partially saturated canthaxanthin on serum antioxidant enzymes of experimental rats1.

Animal group Glutathione reductase Glutathione peroxidase Glutathione transferase Catalase

(lmol/mg protein) (lmol/mg protein) (lmol/mg protein) (lmol/mg protein)
Control 2.771 0.640b 10.821 1.300 3.198 1.667b 0.411 0.068b
Carotenoid (50 ppm) 1.813 0.558ab 9.203 1.673 3.934 1.185b 0.582 0.095a
Carotenoid (100 ppm) 1.538 0.786ab 9.108 1.017 6.767 0.980c 0.526 0.083ab
Carotenoid (250 ppm) 1.208 0.861a 9.294 0.734 12.738 4.025ac 0.541 0.06a
Values are means SD (n = 6). Values in a row not sharing a common superscript are signicantly (P < 0.05) different from each other.

Table 7
Effect of diet containing partially saturated canthaxanthin on hepatic antioxidant enzymes of experimental rats1.

Animal group Glutathione reductase Glutathione peroxidase Glutathione transferase Catalase

(nmol/mg protein) (nmol/mg protein) (nmol/mg protein) (nmol/mg protein)
Control 188.82 27.18 1.991 0.524b 20.10 6.77c 0.708 0.329b
Carotenoid (0.005%) 222.04 17.01 3.811 0.809a 39.30 3.85b 3.519 1.651a
Carotenoid (0.010 5) 217.63 27.39 3.405 1.094ab 39.68 6.51b 2.488 1.337ab
Carotenoid (0.025%) 231.50 49.37 3.785 1.428ab 77.05 13.78a 1.909 0.864ab
Values are means SD (n = 6). Values in a row not sharing a common superscript are signicantly (P < 0.05) different from each other.
3102 A. Kumar et al. / Food and Chemical Toxicology 49 (2011) 30983103

their cellular 3-hydroxy-3-methylglutaryl-CoA reductase activity Research (ICMR) New Delhi, India, for supporting their research
(Fuhrman et al., 1997). Unlike b-carotene or canthaxanthin that through the award of Senior Research Fellowship.
did not affect antioxidant molecules and lipid peroxides of rat plas-
ma or liver (Alam and Alam, 1983; Palozza et al., 2000), PSC in-
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