life sci paper

In vivo antioxidant activity of carotenoids from
Dunaliella salina — a green microalga
K.N. Chidambara Murthy, A. Vanitha, J. Rajesha,
M. Mahadeva Swamy, P.R. Sowmya, Gokare A. Ravishankar*
Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 020, India
Received 8 July 2004; accepted 16 October 2004
Abstract
Dunaliella salina a green marine alga is known for its carotenoid accumulation, having various applications
in the health and nutritional products. The purpose of present study was to evaluate the ability of D. salina
algal powder extract to protect against oxidative stress In vivo using animal models. Treatment of albino Wistar
strain rats with 125 Ag /kg and 250 Ag/kg b.w. showed significant protection when compared to toxin treated
(CCl4) group. Since h-carotene is major constituent of Dunaliella the results were also compared with group
treated with 250 Ag/kg b.w (p.o.) synthetic all trans h-carotene. Treatment of CCl4 at dose of 2.0 g /kg b.w
decreased the activities of various antioxidant enzymes like catalase, superoxide dismutase (SOD) and
peroxidase by 45.9%, 56% and 54% respectively compared to control group and lipid peroxidation value
increased nearly 2 folds. Pretreatment of rats with 125 Ag carotenoid followed by CCl4 treatment caused
restoration of catalase, SOD and peroxidase by 25.24%, 23.75 and 61.15% respectively as compared to control.
The group treated with 250 Ag/kg has shown the restoration of 53.5%, 57.7 and 90.64% of catalase, SOD and
peroxidase, respectively. This group has shown 75.0% restoration of peroxidation compared to control group of
animals. The above enzyme activities were not significantly restored in group treated with synthetic all trans h-
carotene, which showed 7.5%, 23.8% restore in catalase and peroxidase content. The level of superoxide
dismutase remained same and lipid peroxidation value decreased only by 23% in synthetic all trans h-carotene
treated group in comparison with control group. These results clearly indicate the beneficial effect of algal
carotenoid compared to synthetic carotene as antioxidant. Owing to this property, the algae Dunaliella can be
0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2004.10.015
* Corresponding author. Tel.: + 91 821 2516501; fax: +91 821 2517 233.
E-mail address: pcbt@cscftri.ren.nic.in (G.A. Ravishankar).
Life Sciences 76 (2005) 1381–1390
www.elsevier.com/locate/lifescie

further extended to exploit, its possible application for various health benefits as nutraceuticals and food
additive.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity; Carbon tetrachloride; Dunaliella salina; LDL; Carotenoids; SOD
Introduction
There is increasing interest in the use and measurement of antioxidant capacity in food and
pharmaceutical preparations and in clinical studies. The interest is mainly due to the role of reactive
oxygen species (ROS) in aging process and pathogenesis of many diseases in which, ROS are
mainly involved (Cao and Prior, 1998). Many studies have shown that these ROS, including oxygen
free radicals are causitive factors in the etiology of degenerative disorders including some
hepatopathies and other serious organ damage (Ames et al., 1993; Poli, 1993). ROS have also
been shown to modify the damage proteins, carbohydrates and DNA in both In vitro and In vivo
models (Halliwell and Gulteridge, 1990). These free radicals attack unsaturated fatty acids of
biomembrane which results in lipid peroxidation and desaturation of proteins and DNA, which
causes series of deteriorative changes in the biological systems leading to cell inactivation. Thus
identification of antioxidants, which can retard the process of lipid peroxidation by blocking the
generation of free radical chain reaction, has gained importance in recent years (Murthy et al., 2002).
The antioxidants may act by raising the levels of endogenous defense by up regulating the
expression of genes encoding the enzymes such as superoxide dismutase (SOD), catalase, glutathione
peroxidase or lipid peroxidase (Aruoma, 1994; McCord, 1994). According to In vitro and In vivo
studies several classical antioxidants have been shown to protect various cells like hepatocytes,
nephrocytes against lipid peroxidation or inflammation, thereby preventing the occurrence of hepatic
necrosis, kidney damage and other radical associated activities (Halliwell, 1990; Hsio et al., 2003;
Yoshikawa et al., 1996).
Dunaliella salina is a unicellular green alga belonging to family Chlorophyceae. It is known to
accumulate carotenoids under various stress conditions. The algal cells do not contain rigid cell wall,
instead are surrounded by a thin elastic membrane. This alga can yield three major valuable products
namely, glycerol, h-carotene and high protein. In recent years, it is mainly cultivated for carotenoids.
Dunaliella salina under ideal conditions can yield ~400 mg h-carotene/m2
of cultivation area (Finney
et al., 1984).
Apart from being precursor for Vitamin-A synthesis in the body, antioxidant activity of h-carotene
is well known. h-Carotene is an unusual type of antioxidant, which acts by radical trapping; the
activity is optimum at tissue oxygen pressure (Burtan and Ingold, 1984). Epidemiological evidence
have shown h-carotene to prevent cancer of various organs like lungs, stomach, cervix, pancreas,
colon, rectum, breast, prostate and ovary by means of antioxidant activity (Poppel and Goldbohm,
1995). Other than antioxidant property they can influence intracellular communication (Sies and Stahl,
1997), immune response (Hughes et al., 1997), neoplastic transformation and control of growth
(Bertram and Bortkiewicz, 1995). Moreover, carotenoids and their metabolites like retinal,
apocarotenoids, ketones, aldehydes and epoxides influence the biochemical pathways (William et
al., 2000; Yeum and Russel, 2002).
K.N. Chidambara Murthy et al. / Life Sciences 76 (2005) 1381–13901382

Carotenoids quench singlet oxygen primarily by physical mechanism, in which the excess energy of
singlet oxygen is transferred to the carotenoids electron rich structure. These carotenoids get excited by
the added energy into a triplet state, and then relaxes into ground state by loosing the extra energy in the
form of heat. This being a physical phenomenon the structure of carotenoid is unchanged, as a result
carotenoids offers to protect against further singlet oxygen and the process continues on and on. The
reaction rate constant an indicator of the efficacy of a carotenoid as an antioxidant is found to be
relatively less for h-carotene, the same was found to be maximum for lycopene and gamma carotene.
Hence antioxidant activity is better in case of natural carotenoids where they are found in the form of
mixture of several isomers, unlike synthetic ones. Carotenoids fight against free radicals in more than
one mechanism, like by supplying missing electron to the free radicals from other molecules or by
forming adduct with such radicles. In both the cases electron rich nature of carotenoids make them
attractive to radicles, by which they protect lipids, proteins and DNA from radical damage (Di Masico et
al., 1989).
Mokady et al. (1989) have shown that Dunaliella is safe and can be a potential source of food
supplement. Hence present work focused on evaluating the utility of D. salina as a potential source of
antioxidant, and also to study the biological activities of carotenoids of Dunaliella.
Materials and methods
All the solvents/ chemicals used were of analytical/HPLC grade obtained from Merck Mumbai, India,
UV visible spectrum measurements were carried out using Shimadzu 160A Spectrophotometer,
Shimadzu Instrumentation co. USA. Standard h-carotene was obtained from Aldrich (Sigma Chemicals
Co., St.Louis Mo, USA).
Cultivation of Dunaliella
Dunaliella salina 19-3 was obtained from German culture collection-Sammlung Von Algen Kutturen
Universitat Gottingen Germany initially culture was maintained on using AS-100 medium (Vonshak,
1986). Further it was grown in modified Ben Amortz medium (Ben Amortz et al., 1983) comprising of
MgSO4, 7H20 2.5 mM, MgCl2 2.5 mM, CaCl2 0.3 mM, KH2PO4 0.2 mM, KNO3 5.0 mM, NaCl 1.5
mM, NaHCO3 5.0 mM, H3BO3 0.5 mM, CoCl2 6H2O 1.0 mM, MnCl2 4H2O 7 mM, ZnCl2 1.0 mM,
FeCl3 1.5 mM, (NH4)6 Mo7O2 4H2O 1.0 mM (Trace metal mixture). The 14 day old culture was diluted
with equal amount of 2% salt water and subjected for carotenogenesis by exposing to direct sunlight.
After 2 days, biomass was seperated using online centrifuge (M/s Sharples, U.K), The wet biomass was
lyophilized and used for extracting carotenoids using n-Hexane: Iso propyl Alcohol (1:1) and dried
under vacuum. The dried and solvent free carotenoid aliquots were dissolved in olive oil and used for the
study.
HPLC of carotenoids
The total carotenoids were extracted using n-Hexane and isopropyl alcohol (1:1) and estimated
spectrophotometrically by the method of Devis (1976) by measuring absorption at 450 nm. HPLC
system consists of Halwett packard, (Palo, Acto CA) equipped with a quaternary pump fitted with a
K.N. Chidambara Murthy et al. / Life Sciences 76 (2005) 1381–1390 1383

zorbax C18 analytical column (25 cm Â 4.6 nm I.D 5A particle size). The injection system (Rheodyne)
used was 20 Al. Detection was done by an HP 1250 series variable wavelength detector at wavelength of
450.0 nm. The gradient mobile phase consisted of acetonitirile (A) and chloroform (B) with a flow rate
of 1.0 mL/min. The elution program involved a linear gradient from 80 to 20% of A for 0–5 min and B
was 20 to 80% in 5–15 min and again 80% of A for 15–20 min followed by 5 min equilibrium. Total
programme time was of 25 min. The compounds were quantified using HP chemstation software.
Samples were dissolved in mobile phase and 10 Al volume was injected.
Experimental design
Albino rats of either sex of the Wister strain weighing 180–220 gm were used for the studies. The
animals were grouped into five groups each of these were maintained on the prescribed diet for a
period of 15 days. The first group refered to as normal were fed with commercial diet and 1.0 mL of
olive oil dayÀ1
. The second group referred to as control received regular commercial diet administered
with toxin (CCl4 and olive oil 1:1 w/w). The third and fourth groups respectively were fed with
normal diet and supplemented with carotenoids of Dunaliella (125 and 250 Ag. kgÀ1
b.w dayÀ1
).
Fifth group was treated with synthetic h-carotene orally for 14 days at a dose of 250 Ag/kg b.w.
(Dissolved in olive oil). These dose was selected based on the report of Van vliet et al. (1996). The
animals of 3rd to 5th group were administered a single oral dose of CCl4 (1:1 (w/w) in olive oil)
equivalent to 2.0 g/ kg b.w. on 14th day. After 24 hr, animals treated with CCl4 were sacrificed and
liver from each animal was isolated to prepare the liver homogenate i.e. on 15th day and so also the
normal groups were sacrificed on the same day. Liver homogenate 5.0% (w/v) was prepared with 0.15
M KCl and centrifuged at 800 g for 10 min. The cell free supernatant was used for the estimation of
lipid peroxidation, catalase, peroxidase and SOD assay.
Catalase assay
The catalase assay was carried out by the method of Aebi (1984). One millilitre of liver homogenate
from group 1–5 was taken with 1.9 mL of phosphate buffer in different test tubes (125 mM pH 7.4). The
reaction was initiated by the addition of 1 mL of hydrogen peroxide (30 mM). Blank without liver
homogenate was prepared with 2.9 mL of phosphate buffer and 1 mL of hydrogen peroxide. The decrease
in optical density due to decomposition of hydrogen peroxide was measured at the end of 1 min against
the blank at 240 nm. Units of catalase were expressed as the amount of enzyme that decomposes 1 AM
H202 per minute at 258C. The specific activity was expressed in terms of units per milligram of protein.
Estimation of SOD
The assay of SOD was based on the reduction of nitroblue tetrazolium (NBT) to water insoluble blue
formazan, as described by Fedovich (1976). Liver homogenate (0.5 mL) was taken, and 1 mL of 125
mM sodium carbonate, 0.4 mL of 24 AM NBT, and 0.2 mL of 0.1 mM EDTA were added. The reaction
was initiated by adding 0.4 mL of 1 mM hydroxylamine hydrochloride. Zero time absorbance was taken
at 560 nm followed by recording the absorbance after 5 min at 258C. The control was simultaneously run
without liver homogenate. Units of SOD activity were expressed as the amount of enzyme required
inhibiting the reduction of NBT by 50.0%. The specific activity was expressed in terms of units per
milligram of protein.

Estimation of peroxidase
The peroxidase assay was carried out as per the method of Nicholas (1962). Liver homogenate (0.5
mL) was taken and to this were added 1 mL of 10 mM KI solution and 1 mL of 40 mM sodium acetate
solution. The absorbance of potassium periodide was read at 353 nm, which indicates the amount of
peroxidase. Twenty micro liters of hydrogen peroxide (15 mM) was added, and the change in the
absorbance in 5 min was recorded. Units of peroxidase activity were expressed as the amount of enzyme
required to change the 1OD per minute. The specific activity was expressed in terms of units per
Lipid peroxidation activity
Thiobarbituric acid (TBA) reacts with malondialdehyde (MDA) to form a diadduct, a pink
chromogen, which can be detected spectrophotometrically at 532 nm (Buege and Aust, 1978). In this
reaction the TBARS was measured in terms of MDA and expressed as MDA equivalent. Liver
homogenate (0.5 ml) and 1 ml of 0.15 M KCl were taken. Peroxidation was initiated by adding 250 AL
of 0.2 mM ferric chloride. The reaction was run at 378C for 30 min. The reaction was stopped by adding
2 mL of an ice-cold mixture of 0.25 N HCl containing 15% trichloroacetic acid, 0.30% TBA, and 0.05%
BHT and was heated at 808C for 60 min. The samples were cooled and results were expressed as MDA
equivalent, which was calculated by using an extinction coefficient of 1.56 Â 105
MÀ1
cmÀ1
. One unit
of lipid peroxidation activity was defined enzyme required to convert 1 mole of TBA in to TBRS
(measured in terms of MDA equivalent). The specific activity was expressed in terms of units per
Determination of proteins
Protein was determined using the method of Lowry et al. (1951).
Statistical analysis
The experiments were done in triplicate. Data are expressed as mean F SD. One-way analysis of
variance (ANOVA) was used and the test was used for comparison of mean values. All tests were
considered to be statistically significant at p b 0.001.
Results and discussion
The yield of Dunaliella salina was found to be 0.3 g/L on dry wt. basis. The total carotenoid content
was found to be 2.87% w/w. The HPLC chromatogram of Dunaliella carotenoids showed the presence
of h-carotene as the major component along with other carotenoids. Other carotenoids were a-carotene,
lutein, lycopene, were confirmed by UV- absorption maxima. The identities of these peaks were
confirmed by determination of relative retention times and by spiking with standard h-carotene. The
relative percentage of h-carotene was 86.5% (Fig. 1).
Treatment of toxin to Dunaliella carotenoids and synthetic carotenoids treated animals have shown
protection, which was estimated in terms of content of Hepatic enzymes namely, Catalase, Peroxidase,
Super oxide desmutase and Anti lipid peroxidase. Treatments of rats with toxin at 2.0 g/kg body weight
significantly reduced the levels of catalase, peroxidase and SOD by 84.88, 118.11 and 127.16%
respectively (Table 1). On the other hand, lipid peroxidation increased by 3 folds as compared to normal

due to the CCl4 treatment. Among all the groups of experimental animals, the one which was treated
with Dunaliella carotenoids at the concentration of 250 A/kg b.w. showed maximum activity for
protection against CCl4 induced hepatic damage, when compared to control and synthetic carotenoid
Table 1
Effect of carotenoids of Dunaliella and synthetic h-carotene on hepatic enzymes in CCl4 intoxicated group and normal rats
Groups Catalase Peroxidase Superoxide desmutase Anti lipid peroxidase
Normal 597 F 5.98 8.43 F 5.6 16.81 F 3.6 23.12 F 2.8
Control (CCl4 treated) 322.96 F 8.36 3.83 F 3.6 7.4 F 2.8 41.48 F 2.0
Dunaliella carotenoids 125 Ag/kg 647.83 F 5.90 * 13.44 F 5.8 * 17.8 F 3.2 11.51 F 1.4 *
Dunaliella carotenoids 250 Ag/kg 815.52 F 8.95 ** 16.86 F 3.1 ** 20.52 F 1.2 * 5.77 F 0.98 **
Synthetic 100 Ag/kg 604.63 F 10.8 10.21 F 4.0 15.79 F 2.6 17.75 F 1.6
Results are expressed as mean F SEM (n = 6).
* Indicates significant and ** indicates highly significant compared to control group at p b 0.001.
Fig. 1. HPLC chromatograms of carotenoids of different samples A: Standard h-Carotene, B: Dunaliella extracts showing other
carotenoids.

treated group as measured by the levels of hepatic enzymes (Table 1). However, pre-treatment of rats
with 250 Ag of carotenoids (in terms of total carotenoid) preserves catalase, peroxidase and SOD
activities, which are comparable with control values of the enzyme. Restoration of catalase was 183.47
and 131.08% and when compared to toxin treated group, respectively in case of 250 Ag, 125 Ag of
Dunaliella carotenoids whereas same was 98.67% in the animals treated with and 250 Ag synthetic h-
carotene treated group (Table 1). The similar trend was seen in case of peroxidase and SOD enzymes
(Table 1). The protective activity was also observed in the control group, which has shown slightly
higher values of enzymes compared to normal values, which may be due to olive oil. Protocatechuic acid
and elenoic acid derivatives in olive oil are the principles reported to be responsible for the activity
(Masella et al., 1999). They are active against lipid peroxidation, hence the peroxidation in normal group
is also prevented to greater extent. However the influence of the algal carotenoids was much
significantly higher than control and normal group of animals with regards to the activities in other
enzymes like, catalase, peroxidase and SOD.
Protection offered to the animals fed with Dunaliella carotenoids extract is evident from maintenance of
the levels of the liver enzymes even after treatment of the toxin. The lipid peroxidation was restored by 6
folds in case of 250 Ag Dunaliella carotenoid treated group, and the same was restored by 3 - folds in case
of 125 Ag Dunaliella carotenoid treated group. The effect of free radicals on the mean liver detoxification
enzymes (catalase, SOD and peroxidase) reduces the enzyme activity, mainly due to enzyme inactivation
during the catalytic cycle. In these conditions the carotenoids of Dunaliella containing both cis and trans
isomers along with other carotenoids and xanthophyll possibly act as a potent free radical scavenger,
reducing the levels of hydrogen peroxide and superoxideanion and consequently lipid peroxidation and
enzyme inactivation, restoring the enzyme activity. The bioavailability of trans h-carotene is reported to be
3 times more than that of cis, same is found to be more in cis isomer in case of lycopene (Stahl and Sies,
1992; Castenmiller and West, 1998). This may also point towards the possible de novo synthesis of these
enzymes induced by various carotenoids of Dunaliella.
Carbon tetrachloride has been extensively studied as a liver toxicant, and its metabolites such as
trichloromethyl radical (CCl3
S) and trichloromethyl peroxyl radical (CCl3O2
S) are reported to be involved
in the pathogenesis of liver (Recknagel, 1967) and kidney damage. The massive generation of free
radicals in the CCl4 induced liver damage provokes a sharp increase of lipid peroxidation in liver. When
free radical generation is massive, the cytotoxic effect is not localised but can be propagated
intracellularly, increasing the interaction of these radicals with phospholipids structure and induce in
peroxidation process that destroy organ structure (Gil et al., 2000).
Conclusion
h-carotene is a lipid soluble pigment, precursor of fat-soluble Vitamin A, capable of scavenging of
free radicals. In biological system carotenoids are expected to exert most of their antioxidant effects in
lipid environment. Since they are lipophilic in nature (Oshima et al., 1993). Both animal and clinical
studies demonstrated the protective role of h-carotene against oxidation and oxidation mediated diseases
(Levy et al., 2000). These studies generally involved feeding of h-carotene supplemented diet followed
by estimation of plasma concentration of h-carotene or isolation of plasma LDL then induction of
oxidation In vitro by chemicals such as copper chloride, ferric sulphate and measurement of subsequent
lag time for oxidation of LDL. However direct evidence for the antioxidant role of h-carotene In vivo is

rare. Natural h-carotenes have shown to have higher bioavailability compared to synthetic ones (Ben
Amortz and Levy, 1996). Isomers present in the algae have shown to inhibit the LDL-oxidation in
diabetes mellitus patients (Levy et al., 2000).
There are few studies that have examined the effect of h-carotene supplementation In vivo (Paetan et
al., 1997; Astorgs et al., 1994; Albanes, 1999). Lin et al. (1998) have reported that 6.16 A Moles of h-
carotene/day is sufficient for maximum protection of LDL in women. Seddon et al. (1994) have reported
that h-carotene from diet have beneficial effects in prevention of age related macular degeneration.
Researches have shown that plasma concentration of carotenoids has influence on cytochrome P450 and
other key enzymes required for metabolism in the body (Gradelet et al., 1996).
Our present study indicates a protective role for h-carotene rich algae in the reduction of oxidative
stress. Furthermore, it restores the activity of hepatic enzymes like, Catalase, Peroxidase and Super oxide
desmutase, which in turn protects vital organs against xenobiotic and other damages, results have shown
that prevention of lipid oxidation mainly peroxidation to greater extent.
In summary the results indicates that the carotenoids of Dunaliella are capable of enhancing or
maintaining the activity of hepatic enzymes, which are involved in combating ROS. Also the feeding of
the carotenoids provides the protection against toxicity.
Acknowledgement
Authors are grateful to Department of Biotechnology, Government of India for financial assistance to
the project. KNC Murthy would like to acknowledge Council of Scientific and Industrial Research
(CSIR) , Government of India for Senior Research Fellowship.
References
Aebi, H., 1984. Catalase In vitro methods of analysis. Methods in Enzymology 105, 121–125.
Albanes, D., 1999. h-carotene and lung cancer: a case study. American Journal of Clinical Nutrition 69, 1345S–1350S
(Supplement).
Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants and the degenerative diseases of aging. Proceedings
of the National Academy of Sciences of the United States of America 90, 7915–7922.
Aruoma, O.I., 1994. Nutrition and health aspects of free radicals and antioxidants. Food Chemistry 32, 671–683.
Astorg, P., Gradelet, S., Leclerc, J., Canivnc, M.C., Siess, M.H., 1994. Effect of h-carotene and canthaxanthin on liver
xenobiotic metabolizing enzymes in the rats. Food and Chemical Toxicology 32 (8), 735–742.
Ben Amortz, A., Levy, Y., 1996. Bioavailability of natural isomers mixture compared with synthetic all-trans b- carotene in
human serum. American Journal of Clinical Nutrition 63, 729–734.
Ben Amortz, A., Kartz, A., Avron, M., 1983. Accumulation of h-carotene in halotolarent algae: purification and
characterization of h-carotene globules from Dunaliella bardawil. Journal of Phycology 18, 529–537.
Bertram, J.S., Bortkiewicz, H., 1995. Dietary carotenoids in habit neoplastic transformation and modulate gene expression in
mouse and tumor cells. American Journal of Clinical Nutrition 62, 13275–13365.
Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods in Enzymology, 302–310.
Burtan, G.W., Ingold, K.U., 1984. h-carotene an unusual type of lipid antioxidant. Science 224, 569–574.
Cao, G., Prior, R.L., 1998. Comparison of different analytical methods for accessing total antioxidant capacity of human serum.
Clinical Chemistry 44, 1309–1315.
Castenmiller, J.J.M., West, C.E., 1998. Bioavailability and bioconversion of carotenoids. Annual Review of Nutrition 18,
19–38.

Devis, B.M., 1976. Carotenoids: Chemistry and biochemistry of plant pigments, 2nd edition, Vol II. Academic press,
London, pp. 248–249.
Di Masico, P., Kaiser, S., Sies, H., 1989. Lycopene as the most efficient carotenoid singlet oxygen quencher. Biochemical
Society Transactions 274, 532–538.
Fedovich, B.C., 1976. Superoxide dismutase; improved assay and an assay applicable to acrylamide gel. Analytical
Biochemistry 10, 276–287.
Finney, K.F., Pomeranz, Y., Bruinsma, B.L., 1984. Use of algae Dunaliella as a protein supplement in bread. Cereal chemistry
61 (5), 402–406.
Gil, M.I., Tomas-Barberan, F.A., Hess pierce, B., Holecroft, D.M., Kader, A.A., 2000. Antioxidant activity of pomegranate juice
and its relationship with phenolic composition and processing. Journal of Agricultural and Food Chemistry 48, 4581–4589.
Gradelet, S., Astorg, P., Leclerc, J., Chevalier, J., Vernevant, M.F., Siess, M.H., 1996. Effect of canthaxanthin , astaxanthin ,
lycopene and lutein on liver xenobiotic metabolizing enzymes in rats. Xenobiotica 26, 49–63.
Halliwell, B., 1990. How to characterize a biological antioxidant? Free radicals Research Communicatons 9, 1–32.
Halliwell, B., Gulteridge, J.M.C., 1990. Roll of free radicals and catalytic metal ions in human diseases; an overview. Methods
Enzymology 105, 105–114.
Hsio, G., Shen, M.Y., Lin, K.H., Lan, M.H., Wu, Li-Yu, Chou, D.S., Lin, C.H., Su, C.H., Sheu, J.R., 2003. Antioxidant &
hepatoprotective effects of Anthodia canphore extracts. Journal of Agricultural and Food Chemistry 512, 3302–3308.
Hughes, D.A., Wright, A.J.A., Finglas, P.M., Peerless, A.C.J., Bailey, A.L., Astley, S.B., Pindar, A.C., Southon, S., 1997. The
effect of h-carotene supplementation on the immune function of blood monocytes from healthy male non smokers. Journal
of Laboratory and Clinical Medicine 129, 309–317.
Levy, Y., Zaltsberg, H., Ben Amortz, A., Kantery, Y., Aviram, M., 2000. Dietary supplementation of natural isomer mixture of
LDL derivatived from patients with diabetes mellitus. Annals of Nutrition and Metabolism 44 (2), 54–60.
Lin, Y., Burri, B.J., Neidlinger, T.R., Muller, G.H., Dueker, S.R., Clifford, A.J., 1998. Estimating the concentration of h-
carotene required for the maximal protection of LDL lipoproteins in women. American Journal of Clinical Nutrition 67,
837–845.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, J.L., 1951. Protein measurement with Folin-phenol reagent. Journal of
Biological Chemistry 193, 265–275.
McCord, J.M., 1994. Free radicals and pro-oxidants in health and nutrition. Food Technologist 48, 106–110.
Masella, R., Cantafora, A., Modesti, D., Cardilli, A., Gennaro, L., Bocca, A., Coni, E., 1999. Antioxidant activity of 3,4-DHPEA-
EA and protocatechuic acid: a comparative assessment with other olive oil biphenols. Redox Report 4 (3), 113–121.
Mokady, S., Abramovici, A., Co gau, U., 1989. The safety evaluation of Dunaliella bardawil as a potential food supplement.
Food and Chemical Toxicology 4, 221–226.
Murthy, K.N.C., Jayaprakasha, G.K., Singh, R.P., 2002. Studies on Antioxidant activity of Pomegranate (Punica granatum)
peel Extarcts Using In vivo Models. Journal of Agricultural and Food Chemistry 50, 4791–4795.
Nicholas, M.A., 1962. A spectrophotometric assay for iodide oxidation by thyroid peroxidase. Analytical Biochemistry 4,
311–345.
Oshima, S., Ojima, F., Sakamoto, H., Ishigauro, Y., Terao, J., 1993. Inhibitory effect of h-carotene and astaxanthin on
photosensitized oxidation of phospholipid bilayer. Journal of Nutritional Science and Vitaminology 39, 607–615.
Paetan, I., Chen, H., Goh, M.N., White, W.S., 1997. Interactions in the postprandial appearance of h-carotene and
canthaxanthin in plasma triglycerol rich lipoproteins in human. American Journal of Clinical Nutrition 66, 1133–1143.
Poli, G., 1993. Liver damage due to free radicals. British Medical Bulletin 49 (3), 604–620.
Poppel, G.V., Goldbohm, R.A., 1995. Epidemiological evidences for h-carotene and cancer prevention. American Journal of
Clinical Nutrition 62, 1393S–1402S.
Recknagel, R.O., 1967. Carbon tetrachloride hepatotoxicity. Pharmacological Reviews 19, 145–208.
Seddon, J.M., Ajani, U.A., Sperduto, R.D., Hiller, R., Blair, N., Burton, T.C., Farber, M.D., Gragoudas, E.S., Haller, J., Miller,
D.T., 1994. Dietary carotenoids, Vitamin C and E and advantages related macular degeneration. JAMA 272, 1413–1420.
Sies, H., Stahl, W., 1997. Carotenoid and intracellular communications via gap junction. International Journal of Vitamin
and Nutrition Research 67, 364–367.
Stahl, W., Sies, H., 1992. Uptake of lycopene and its geometrical isomer is greater from heat-processed tomato juice in humans.
Journal of Nutrition 122, 2131–2166.
Van vliet, T., Van vilssingen, M.F., Van schaik, F., Van den Berg, H., 1996. Beta carotene absorption and cleavage in rats is
affected by the vitamin A concentration of the diet. Journal of Nutrition 126 (2), 499–508.

Vonshak, A., 1986. Laboratory techniques for cultivation of Micro algae. CRC handbook of micro algal mass culture. CPC
press, Florida, pp. 345–349.
William, A., Pryor, B.S., Wilhelm, S., Cheryl, L., Rock, R.D., 2000. h-carotene from Biochemistry to clinical trials. Nutrition
Review 1, 39–53.
Yeum, K.J., Russel, R.M., 2002. Carotenoids bioavailability & bioconversion. Annual Review of Nutrition 22, 483–1254.
Yoshikawa, T., Furukawa, Y., Musakoun, M., Takumana, S., Kondo, M., 1996. Effect of Vitamin E on D. galactosamine
induced or CCl4 induced hepatotoxicity. Digestion 25, 222–229.

life sci paper

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (20)

Similar to life sci paper

Similar to life sci paper (20)

life sci paper