1. Hepatology Research 33 (2005) 313–319
Protective effect of Dunaliella salina—A marine micro alga, against
carbon tetrachloride-induced hepatotoxicity in rats
K.N. Chidambara Murthya, J Rajeshab, A. Vanithaa,
M. Mahadeva Swamya, G.A. Ravishankara,∗
a Plant Cell Biotechnology Department, Central Food Technological Research Institute, Cheluvamba Mansion, Mysore 570 020, Karnataka, India
b Department of Biochemistry, Manasagangothri, University of Mysore, Mysore 570 006, Karnataka, India
Received 25 October 2004; received in revised form 11 January 2005; accepted 26 August 2005
Abstract
This is the first report on the hepatoprotective potentials of marine micro algae Dunaliella species. Dunaliella salina, halotolarent green
alga was cultivated in modified autotrophic medium. The alga was subjected to light and nutrient stress in order to accumulate (␤-carotene
along with other carotenoids. Such ␤-carotene enriched yellow cells were fed to rats by mixing with regular feed at the dose of 2.5 and
of 5.0 g kg−1
b.w. for 2 weeks. The degree of hepatoprotection was measured up on challenging animals with toxin (2.0 g kg−1
of carbon
tetrachloride) by estimation of biochemical parameters like, serum transaminases (serum aspartate transaminase (S)AST and serum alanine
transaminase (S)ALT), serum alkaline phosphatase and total protein. The results were compared to animals on normal diet and with group fed
with 100 ␮g kg−1
b.w. of standard all trans ␤-carotene. Among the three test groups the group fed with algae of 5.0 g kg−1
body weight, showed
maximum protection. The levels of (S)AST and (S)ALT was found to be 61.3 ± 6.4 and 80.7 ± 5.6%, against 90.8 ± 10.5 and 144.7 ± 13.9%
in case of standard ␤-carotene. The protein contents were increased in case of control to 6.1 ± 0.7 and the same was found to be significantly
less in case of 5.0 g kg−1
Dunaliella fed group, which shown 5.6 ± 0.8% total protein. However, the activity of 2.5 g kg−1
was also significant
comparatively (P < 0.05). The results indicate that Dunaliella, which contains isomeric forms of ␤-carotene can act as good antihepatotoxic
when compared to synthetic all trans ␤-carotene. Dunaliella has shown the presence of both cis and trans isomeric forms of ␤-carotene,
where as synthetic compounds contain only trans isomer. Hepatoprotectivity may be due to presence of various isomeric forms of carotene
and other oxygenated carotenoids (xanthophylls) in algae.
© 2005 Published by Elsevier Ireland Ltd.
Keywords: Dunaliella salina; Hepatoprotective activity; ␤-Carotene; Carbon tetrachloride
1. Introduction
Modern medicinal research has led to various break-
throughs by the discovery of drugs to cure and prevent chal-
lenging ailments and disorders via biotechnology. However,
there are still some problems, which have not got clear solu-
tion i.e. a single, or treatment technique, one such is liver
Abbreviations: (S)AST, Serum aspartate transaminase; (S)ALT, Serum
alanine transaminase; SALP, Serum alkaline phosphatase; CCl4, Carbon
tetrachloride
∗ Corresponding author. Tel.: +91 821 2516 501; fax: +91 821 2517 233.
E-mail address: pcbt@cscftri.res.nic.in (G.A. Ravishankar).
disorder in particular liver damage. The liver is one of the vital
organ, susceptible for repeated damage due to its functions.
Apart from alcohol other damage causing agents include
antibiotics and their metabolites, other drugs used for vari-
ous diseases and infections will significantly contribute to the
process of damage [1]. In present market, most of the exist-
ing hepatoprotectives are polyherbal formulations containing
more than 5–6 herbal extracts, basically containing antioxi-
dant principles [2]. However, management of liver damage
by a single or precise medicament is still a leading challenge
in this research area. Hence, there is urge for finding newer,
precise and potential sources of hepatoprotective agents from
natural sources.
1386-6346/$ – see front matter © 2005 Published by Elsevier Ireland Ltd.
doi:10.1016/j.hepres.2005.08.008

2. 314 K.N. Chidambara Murthy et al. / Hepatology Research 33 (2005) 313–319
Algae are unexploited sources due to their limited distri-
bution in natural habitat and less information on conditions
for growth and utilization. However, in recent years these
algae are gaining importance due to nutritional composition
and various bioactive compounds they produce to accustom
to the biodiversity of marine environment. Some of the bio-
logically significant compounds of algal origin include car-
rageenan, sulpholipids, pigments like phycocynain and so on
[3].
The genus Dunaliella includes halotolarent, unicellular,
motile green algae with exceptional morphological and phys-
iological properties [4]. Dunaliella devoid of rigid cell wall
and contains a single, large cup shaped chloroplast [5] and
can accumulate massive amount of ␤-carotene, primarily in
response to high light intensity [6]. This makes it interest-
ing subject for utilization as a source of ␤-carotene, which
is well known a precursor of vitamin A, antioxidant and
also a bioactive molecule. Apart from ␤-carotene it is also a
source of protein that has good utility value, rich in essential
fatty acids and it is safe to utilize directly in food formu-
lations [7]. Dunaliella species have shown to exhibit vari-
ous biological activities, like anti-hypertensive, bronchodila-
tor, analgesic and muscle relaxant and anti-edema activity
[8].
The present pharmacological investigation focuses on
evaluation of the efficacy of Dunaliella whole cells for pos-
sible protection against CCl4-induced hepatotoxicity in rats.
Carbon tetrachloride induces hepatic injury characterized by
leakage of cellular enzymes into the blood stream by centri-
olobular necrosis [9]. The degree of protection was measured
by using biochemical markers like, serum AST, ALT, ALP
and total protein. The results of the same were compared
with synthetic ␤-carotene, as it is the major constituent of the
algae.
2. Materials and methods
2.1. Chemicals
SALP, (S)ALT, (S)AST kits were purchased from Span
Diagnostics Ltd., India. All the solvents and chemicals used
for experiment were of analytical grade, solvents used for
HPLC were of HPLC grade and purchased from Ranbaxy fine
chemicals Ltd., India. Standard ␤-carotene was from Sigma
Chemicals Ltd., USA.
2.2. Algal biomass
Dunaliella salina (No. 19-3) obtained from Sammulung
von Algen kulturen, Pflanzen, Physiologische Institute, Uni-
versitat Gottingen, Gottingen, Germany. The organism was
initially maintained in AS-100 medium [10], further it was
cultivated using modified medium [11], with slight modifica-
tion for better yield of biomass and ␤-carotene accumulation.
Carotenogenesis was induced by high light (25–30 klx) by
exposing cultures to direct sunlight after the growth for 14
days for 3 days period. The yellowish biomass was har-
vested by centrifugation and lyophilized to remove water.
This lyophilized biomass of carotenoid accumulated cell was
used for nutritional analysis and formulation of feed for eval-
uation of the activity.
2.3. Formulation of feed
Known proportion of the dried algae powder was mixed
with powdered standard commercial feed (Brook Bond India
Pvt. Ltd.) and reconstituted as that of commercial pellet feed
by vacuum drying. This was fed to animals according to the
dosage profile. Carotenoid content was also measured in feed.
It was found that, there was no loss of carotenoids during the
process of formulation of feed.
2.4. Analysis of nutritional composition
Yellow biomass was subjected to biochemical analysis
of total protein [12], carbohydrate by phenol–sulfuric acid
method [13], total lipids by AOAC method [14] and fatty
acid by Levy et al. method [15]. HPLC was done for both
crude and a fraction after passing through column 12.5 mm
(i.d.) × 20 cm glass column containing diatomaceous earth
and silica gel G (1:1) using varying concentration of n-
Hexane and acetone in order to remove chlorophyll [16], ␤-
carotene was also estimated by AOAC method and further
quantified by HPLC (LC10A Shimadzu), using ODS (Bonda
pack)Columnwith,isocraticelutionofmethanolandacetoni-
trile (9:1) at a flow rate of l.0 mL/min, at 450 nm wavelength
in UV–vis detector [17].
2.5. Experimental animals
Albino rats of either sex of the Wister strain weigh-
ing 180–220 g were used for the studies. The animals were
grouped into five groups each group consisting of six ani-
mals each (n = 6). The first group served with normal diet
without treatment of toxin, the second group named as control
received normal diet and was administered with toxin (CCU).
The third and fourth groups were treated with Dunaliella
(2.5 and 5.0 g kg−1 which approximately equivalent to 70 and
140 mg of total carotenoids/kg). Fifth group was treated with
synthetic ␤-carotene orally at dose of 100 ␮g kg−1 (dissolved
in olive oil) for 14 days. The animals of 1st–4th groups were
simultaneously administered with olive oil until 14 days. The
animals of 2nd–5th group were given a single oral dose of
CCl4 (1:1 in olive oil) at dose of 2.0 g kg−1 b.w., 6 h after
the last dose of administration of carotene/olive oil at the
14th day. After 24 h, animals were sacrificed and blood was
collected in heparinized tubes from each animal by cardiac
puncture (∼3.0 mL). Serum was separated by centrifugation
and used for the biochemical analysis. Biochemical analysis
for protein, (S)AST, (S)ALT and SALP was done for all the
animals on 15th day.

3. K.N. Chidambara Murthy et al. / Hepatology Research 33 (2005) 313–319 315
2.6. CCl4-induced hepatotoxicity
One of the groups (V) received synthetic ␤-carotene
(100 mg kg−1 dissolved in olive oil) orally. Animals of test
group received diet with algae, these test groups also received
same dose of vehicle i.e. olive oil in order to know the effect
of olive oil on the hepatoprotective activity. Twenty-four
hours after the last day CCl4 was administered at the dose
of 2.0 g kg−1 b.w. (mixed in the ratio 1:1 with olive oil) and
24 h after the CCl4 administration the biochemical parame-
ters were analysed.
2.7. Assessment of liver function
Rats of the entire group were anesthetized by diethyl ether,
24 h after the hepatotoxin administration. The blood was
obtained from by cardiac puncture from dissected animals
and were allowed to clot for 45 min at room temperature.
Serum was separated by centrifugation at 2500 rpm at 30 ◦C
for 15 min and analyzed for various biochemical parameters.
Serum transaminase viz. aspartate transaminase [18], Serum
alanine transaminase [17], alkaline phosphatase [19] were
estimated by using standard enzymatic kits. Total protein was
estimated by Lowry et al. method [20].
2.8. Statistical analysis
Results of the biochemical estimations are reported as
mean ± S.E.M. Total variation, present in a set of data was
estimated by one-way analysis of variance (ANOVA), results
were analysed by tukey’s multiple comparison test using
PRISM 4.0 version software.
3. Results
As shown in Fig. 1a, growth rate of Dunaliella salina was
linear till 14 days and the cells were diluted to get a cell
count of 50 × 104 concentration and exposed to direct sun-
light (25–30 klx intensity) for accumulation of (␤-carotene
(Fig. 1b). The cells turned to yellow colour on 3rd day
of exposure and the same was subjected to harvesting by
centrifugation to get a yield of 0.656 ± 0.65 g L−1 of dry
biomass.
The nutritional composition of yellow biomass, showed
the presence of protein 21.5 ± 1.75% essential fatty acids
i.e. 0.79 ± 0.36%, the ␤-carotene content 2.8 ± 0.27%
w/w (Table 1). HPLC has shown ␤-carotene peak at
9.84 ± 0.15 min and the total concentration was found to be
2.8% w/w and has shown the presence of chlorophyll and
other carotenoids in Dunaliella extracts. The standard and
extract ␤-carotene chromatograms are shown in Fig. 2a and
b.
As shown in Figs. 3–6, activities of the AST (S), ALT
(S), and SALP were markedly elevated while total protein
content decreased in control rats compared to normal rats.
Fig. 1. (a) Growth curve of Dunaliella salina in modified medium. The
graph shows the growth pattern of cells under low light for 14 days in which
there is multiplication of green cells in vegetative phase. (b) Carotene accu-
mulation in Dunaliella salina after exposing to high light stress. The cells
were transferred in to high light by diluting with 2.0% w/v sodium chloride
for carotenogenesis, during which there was accumulation of carotenoids up
to 2.8% w/w.
Administration of Dunaliella at two different doses markedly
prevented CCU induced elevation of (S)AST, (S)ALT, SALP
and diminution of total protein.
Dunaliella salina feeding at 2.5 and 5.0 g kg−1 b.w
decreased (S)ALT by 81.0 ± 13.66, 61.33 ± 6.43%, (S)AST
by 117.93 ± 10.91, 80.66 ± 5.64%, respectively. However,
there was not considerably decrease in case of synthetic
␤-carotene, which was found to be 90.83 ± 10.49 and
144.66 ± 3.84%, respectively. Similarly the alkaline phos-
phatase level was higher in case of CCl4 toxin-treated, where
Table 1
Nutritional composition of Dunaliella salina
Nutritional Component % Value ± S.D
Protein 21.50 ± 1.75
Carbohydrate 26.70 ± 1.11
Total fat 7.78 ± 0.38
Essential fatty acid 0.79 ± 0.36
Total carotenoids 2.80 ± 0.27
␤-Carotene 2.57 ± 0.66

4. 316 K.N. Chidambara Murthy et al. / Hepatology Research 33 (2005) 313–319
Fig. 2. (a) HPLC Chromatogram of standard synthetic all trans ␤-carotene,
(b) HPLC chromatogram of carotenoids of Dunaliella salina crude extract.
This chromatogram shows the presence of other carotenoids like, xantho-
phylls, lutein and these are totally identified as total carotenoids by extinction
coefficient at 450 nm. Peak at 9.841 corresponds to ␤-carotene of Dunaliella,
which constitutes around 80% of total carotenoids.
as it was nearly half in case of 2.5, 5.0 g kg−1 group. Synthetic
␤-carotene-treated group animals have also shown decrease
in the SALP, which was not significant. The total protein con-
tent decreased to 3.90 ± 0.62% in case of control and it was
found to be as high as 8.11 ± 0.68 and 7.8 ± 0.59 in case of
2.5, 5.0 g kg−1 biomass-treated and the same was 5.59 ± 0.77
in case of synthetic ␤-carotene-treated group of animals.
Histology has shown in case of control, hepatocytes with
normal architecture and portal triad, portal veins, hepatic
artery and vein are visible (Fig. 7). However, control group
showed total loss of hepatic architecture, areas of necrosis.
Fig. 3. Effect of Dunaliella biomass and synthetic ␤-carotene on serum
alanine transaminase in Wister albino rats as on 14th day of different animals
in comparison with control and treated groups.
Fig. 4. Effect of Dunaliella biomass and synthetic ␤-carotene on serum
aspartate transaminase activity of serum in Wister albino rats as on 14th day
of different animals in comparison with control and treated groups.
Fig. 5. Effect of Dunaliella biomass and synthetic ␤-carotene on serum
alkaline phosphatase in Wister albino rats as on 14th day of different animals
in comparison with control and treated groups.
Fig.6. EffectofDunaliellabiomassandsynthetic␤-caroteneontotalprotein
content of serum in Wister albino rats as on 14th day of different animals in
comparison with control and treated groups.

5. K.N. Chidambara Murthy et al. / Hepatology Research 33 (2005) 313–319 317
Fig. 7. Histology of liver of different treated groups showing the damage in case of control and the same has been protected in treatment groups (100×).
In case of Dunaliella treated group followed by CCl4, the
liver has retained the normal hepatic architecture with minor
hemorrhage in case of 2.5 g kg−1 treated group and the same
was absent in case of 5.0 g kg−1 treated group.
4. Discussion and conclusion
Carbon tetrachloride induces fatty liver and cell necrosis
and plays a significant role in inducing triacylglycerol accu-
mulation, depletion of GSH, increased lipid peroxidation,
membrane damage and depression of protein synthesis and
loss of enzymatic activity [21,22]. This is due to metabolites
of carbon tetrachloride like, trichloromethyl radical (CCl3
•)
and trichloromethyl peroxyl radical (CCl3O2
•) which are
formed due to the cleavage of carbon-chloride bond (C Cl
bond), these will provoke a sharp increase in the lipid perox-
idation in liver, which also influences the markers release to
plasma [9]. The disturbance in the transport function of the
hepatocytes as a result of hepatic injury causes the leakage
of enzymes from cells due to altered permeability of mem-
brane. This results in decreased levels of (S)AST, (S)ALT
and serum alkaline phosphates in the hepatic cells and a
raised level in serum [23]. Prevention of liver damage by
CCl4 has been widely used as an indicator of liver protec-
tion of drugs in general. Since the changes associated with
CCl4-induced liver damage are similar to that of acute viral
hepatitis, CCl4-mediated hepatotoxicity was chosen as the
experimental model. It has been established that CCl4 is
accumulated in hepatic parenchyma cells and metabolically
activated by cytochrome P450-dependent monooxygenases
to form a trichloromethyl radical (CCl3). The CCl3 radical
alkylates cellular proteins and other macromolecules with
a simultaneous attack on polyunsaturated fatty acids, in the
presence of oxygen, to produce lipid peroxides, leading to
liver damage. Thus, antioxidant or free radical generation
inhibition is important in protection against CCl4-induced
liver lesions [24].

6. 318 K.N. Chidambara Murthy et al. / Hepatology Research 33 (2005) 313–319
Being cytoplasamic in location the damage marker
enzymes like, AST, ALT and ALP are released in serum
[25,26]. These carotenoids show their protective activity by
impairment of CCl4-mediated lipid peroxidation, either by
decrease in production of free radical derivatives or by the
antioxidant activity of these compounds.
It has been therefore found that the different concentra-
tions of D. salina have varied degree of hepatoprotective
activity. Since Dunaliella is reported to be safe for con-
sumption as reported by studies using animals [27], it can
be possibly utilized for such preparations requiring antihep-
atotoxic activity.
Results of above studies showed that the D. salina supple-
mentation in different concentration containing ␤-carotene
along with valuable nutritional factors, like protein and essen-
tial fatty acids, produces characteristic effect on hepatotoxi-
city. ␤-Carotene is responsible for the activity since the same
is known to be antioxidant, which is one of the mechanisms
for hepatoprotective activity [28]. Carrot has shown signifi-
cant hepatoprotection property in rats, which is attributed to
carotenoids it contains [29].
However, Dunaliella contains many carotenoids and xan-
thophylls, which can contribute to greater extent for the same
property.Manycompoundsareknowntobebeneficialagainst
carbon tetrachloride-induced liver injury. They exert protec-
tive action by toxin mediated lipid peroxidation either by
decrease in the production of carbon tetrachloride-derived
free radicals, like trichloromethyl radical and trichloromethyl
peroxyl radical or by their antioxidant potentials. Since
carotenoids are well known for their antioxidant properties it
must be the major mechanism of action by which they protect
liver damage. They are also known for their proton donating
ability hence this may also take care of carbon tetrachloride-
derived free radicals [30].
When the free radical generation is massive, in the CCl4
toxicity, the cytotoxic effect is not localized but can be prop-
agated intracellularly, increasing in the interaction of these
radicals with phospholipids structures and inducing peroxi-
dation process that destroys organ structure. Most of the time
the hepatoprotective activity is most of the time associated
with antioxidant activity, since it is free radical mediated
damage [31]. ␤-Carotene is well known for its antioxidant
properties by various mechanisms, which make Dunaliella
a potent hepatoprotector as it is rich in carotenoids of wide
range.
In contrast to the toxic activation of CCl4 via the P450
2E1 pathway, the detoxification pathway involves GSH con-
jugation of the trichloromethyl radical, a P450 2E1-mediated
CCl4 metabolite. Previous studies on the mechanism of CCl4-
induced hepatotoxicity have shown that GSH plays a key
role in detoxifying the reactive toxic metabolites of CCl4
and that liver necrosis begins when the GSH stores are
markedly depleted. GSH is largely mediated through the
activity of glutathione-S-transferase, and forms adducts with
the toxic metabolites of CCl4. Moreover, GSH contribute to
the detoxification of CCU, and it has been suggested that
one of the principal causes of CCl4-induced liver injury
is lipid peroxidation caused by its free radical derivatives
[32].
Hepatoprotective effect of Dunaliella reported here has
large implications in biotechnological exploitation of these
algae for health food and therapeutic properties, besides its
utility of the algae as a source of (␤-carotene and other
carotenoids as well as other bioactive carotenoids.
Acknowledgements
The authors are thankful to Department of Biotechnol-
ogy, Government of India, for the financial support for the
project and Department of Zoology, University of Mysore,
for providing animal house facility. KNC Murthy is thank-
ful to CSIR, Govt, of India, for providing Senior Research
fellowship.
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