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DOI 10.11603/IJMMR.2413-6077.2018.1.8979

cARRAGEENAN INdUcEs cELL dEATh IN RATs BLOOd

O. M. Kopanytsia, M. I. Marushchak, I. Ya. Krynytska
I. HORBACHEVSKy TERNOPIL STATE MEDICAL UNIVERSITy, TERNOPIL, UKRAINE

Background. Because of its physical and chemical properties, carrageenan is fairly widely used. About 70 % 
of the carrageenan produced in the world is used in the food industry. Previous studies point to the development 
of oxidative stress in rats, by means of which carrageenan chronic enterocolitis was modeled.

Objective. The aim of our study was to investigate the level of apoptosis and necrosis in the suspension of 
leukocytes in rats using 0.5 % and 1.0 % solutions of carrageenan.

Methods. Annexin V (V) binding assays were performed using Annexin V Apoptosis Kit (Sigma Aldrich, USA), 
caspase rate in leukocyte-lymphocyte blood fractions was determined by spectrofotometry.

Results. It was established that in the experimental application of carrageenan, the percentage of leukocytes 
with signs of apoptosis in both experimental groups statistically significantly increased. It was detected by the 
increased activity of effector caspase-3 in 1 month after the experiment in 1.5 times in the 2nd group and in 2.8 
times in the 3rd group vs control data that point to caspase-dependent apoptotic pathway in case of carrageenan 
usage in rats.

Conclusions. Oral use of carrageenan in rats was accompanied by the increase in the number of leukocytes 
with signs of apoptosis. The animals that consumed 1.0 % solution of carrageenan had more obvious increase 
in the activity of caspase-3 in serum relative to a group of rats consuming 0.5 % of carrageenan, proving the 
increase in the severity of apoptotic processes in intestine with the increase of the dose of carrageenan.

KeY WoRdS: carrageenan, apoptosis, caspase-3, rat.

Introduction 
In world practice it is allowed to use about 

500 different substances as nutritional 
supplements, not taking into account a huge 
number of different types of flavors and some 
varieties of combined supplements [1]. 

In the USA, the amount of nutritional 
supplements (nS) exceeds 1500, in the eu it 
reaches 1200, in Russia it is 415, in the Federal 
Republic of Germany – 350 [2]. By the beginning 
of 90s of the 20th century the use of nutritional 
s u p p l e m e n t s  i n  U k ra i n e  w a s  l i m i t e d  i n 
comparison with other countries of Europe and 
the USA [3], only 194 NS were permitted until 
1994, and according to the Cabinet of Ministers 
Resolution in 2000, 221 NS were permitted. In 
addition, in the EU countries it is allowed to use 
more than 400 flavors in food production.

For food and in medical practice, there is a 
nutritional supplement called carrageen that is 
emitted by water extraction from marine algae 
[4]. This NS (E407) constitutes a family of 
polysaccharides (also known as Irish moss) 

contained in the red seaweed Chondrus Crispus, 
Eucheuma species, Gigartina species, and 
others. By chemical nature carrageenan is close 
to agarose and represents unbranched sulfated 
heteroglycans which molecules are made of 
residues of derivatives of D-galactopyranose 
with a strict alternation of α- (1,3) and β- (1,4) 
b o n d s  b e t w e e n  t h e m ,  i . e .  o f  re p e t i t i v e 
disaccharide units including residues of β-d-
g a l a c t o p y r a n o s e  a n d  3 , 6 - a n h y d r o - α - d -
galactopyranose. Depending on the peculiarities 
of the structure of disaccharide repeating units, 
there are three main types of carrageenans, for 
which the letters of the Greek alphabet are used 
to define [1, 5, 6].

Because of its physical and chemical 
properties, carrageenan is a fairly widely used. 
About 70 % of the carrageenan produced in the 
world is used in the food industry: in the 
production of dairy products (chocolate milk, 
sherbets, farmer cheese, cheese paste, baby 
food), meat and fish products (canned food, 
sausage casings, jelly coatings), seasonings, 
nonalcoholic beverages, bakery (bread dough, 
fruit muffins, sugar icing) and confectionery [7].

Previous studies point to the development 
of oxidative stress in rats, by which carrageenan 
chronic enterocolitis was modeled [8]. The 

International Journal of Medicine and Medical Research 
2018, volume 4, Issue 1, p. 67-70
copyright © 2018, TSMU, All Rights Reserved

Corresponding author: Mariya Marushchak, Professor, Depart-
ment of Functional and Laboratory Diagnostics, I. Hor-
bachevsky Ternopil State Medical University, 1 Maydan Voli, 
Ternopil, 46000, Ukraine 
E-mail: marushchak@tdmu.edu.ua
Phone number: +380979981202

O. M. Kopanytsia et al.



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iSSn 2413-6077. iJmmR 2018 Vol. 4 issue 1

activation of lipid peroxidation is caused by 
direct stimulation of generation of active forms 
of oxygen by carrageenan, or indirectly, via the 
tumor-alpha necrosis factor [9]. 

the fundamental of carrageenan influence 
on the body is the development of oxidative 
stress as one of the mechanisms of damage of 
intestine as well as the major multiple organ 
lesions in heart, lungs and liver. Therefore, the 
aim of our study was to investigate the level of 
apoptosis and necrosis in the suspension of 
leukocytes in rats using 0.5 % and 1.0 % 
solutions of carrageenan.

Methods 
The study was conducted on 36 mature 

white nonlinear male rats, which were kept on 
a standard diet at the vivarium of I. Horbachevsky 
Ternopil State Medical University. During the 
study we followed the principles of the 
European Convention for the Protection of 
Vertebrate animals used for experimental and 
other Scientific Purposes (Strasbourg, 1986). 
the rats were divided into 1 control and 2 expe-
rimental groups: the 1st group – control (intact 
animals); the 2nd group comprised the animals 
that consumed 0.5 % solution of carrageenan, 
the 3rd group consisted of the animals that 
consumed 1.0 % solution of carrageenan. The 
2nd and the 3rd groups of animals were provided 
with free access to 0.5 % solution of carrageenan 
and 1.0 % solution of carrageenan in drinking 
water for 1 month [10, 11].

a n n e x i n  V  ( V )  b i n d i n g  a s s a y s  w e r e 
performed using annexin V apoptosis Kit 
(Sigma Aldrich, USA). Apoptotic cells of blood 
leukocyte suspension were identified by flow 
cytometry using flow cytometer Epics xl 
(Beckman Coulter, USA). To distinguish cells 
that had lost membrane integrity, propidium 
iodide (Pi) was added to a final concentration 
of 10 mg/ml before the analysis. The results 
were presented as a percentage of the total 
number of cells as follows: live cells – not 
stained (v -/PI -), cells with early signs of 
apoptosis – stained with annexin (V+/PI-), cells 
with late signs of apoptosis – positive double 
fluorescence staining, cells with signs of 
necrosis – stained with propidium iodide (v-/
PI+).

To determine caspase rate in leukocyte-
lymphocyte blood fractions, 0.25 ml of buffer 
and 50 mcl of 2 mM DEvD-p-NA was added to 
0.7 ml of the test liquid. It was incubated for 
2 hours at 37 °C; the intensity of light absorbance 
was measured at 405 Nm, which was directly 

proportional to the product of hydrolysis of 
Acetyl-Asp-Glu-val-Asp n-nitroanilide caspase – 
3-n-nitroanilide [12].

Statistical analysis 
The results were analyzed using Statistica 

7.0 software and presented as mean with 
standard error of mean. The differences 
between all groups were determined using 
one-way ANOvA, followed by post hoc the least 
Significant difference test. a p-value <0.05 was 
considered statistically significant. 

Results 
it was established that with the experimental 

application of carrageenan, the percentage of 
leukocytes with signs of apoptosis in both 
experimental groups increased significantly 
(Table 1). Thus, the percentage of v+/PI--cells in 
the 2nd group increased in 1.9 times, and in 
group 3 – in 2.2 times vs the control indexes 
(p<0.001). The percentage of leukocytes with 
later signs of apoptosis increased significantly, 
with respect to control: in group 2 – in 8.9 times, 
in group 3 – in 22.3 times (p<0.001). It should 
be noted that the level of necrotic cells when 
introducing 0.5 % ca rra geena n did not 
significantly differ from the normal indices, 
while the use of 1.0 % solution of carrageenan 
in drinking water caused the increase of v-/PI+ 
-cell in 1.7 times (p<0.001).

С a s p a s e s  i n  g e n e r a l  a r e  i m p o r t a n t 
mediators in apoptosis, especially caspase-3, 
which is the main caspase effector that cleaves 
cell substrates. It was established that the 
activity of caspase-3 effector in 1 month of the 
experiment increased in 1.5 times in the 2nd 
group and in 2.8 times in the 3rd group vs control 
data that proved the caspase-dependent 
apoptotic pathway in case of carrageenan use 
for rats (Fig. 1). 

Discussion 
Caspase-3 is probably the best understood 

of the mammalian caspases in terms of its 
specificity and roles in apoptosis. overall, recent 
progress has generally confirmed the notion of 
multiple, complex death pathways (some of 
which require caspase-3 in specific cell types) 
that converge on common events including cell 
shrinkage, blebbing, chromatin condensation 
and DNA. Two established ways of apoptosis 
include internal or mitochondrial, involving 
protein family Bcl–2, cytochrome С and caspase 
– 9 and external with the activation of caspase–8
linking a specific cell receptor fas– and soluble 
tumor necrosis factor receptors on the cell 

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Table 1. Indicators of cell death in serum of the rats in experimental use of carrageenan Me (Q25-Q75)

index Control 2nd group 3rd група
Alive leukocytes, % 95.76 

(95.28; 96.77)
90.05* 

(89.26; 90.43)
83.87*#

(82.62; 86.11)
leukocytes with early signs of apoptosis, % 3.05 

(2.20; 3.68)
5.89* 

(5.55; 6.20)
6.73*#

(5.83; 7.55) 
leukocytes with late signs of apoptosis, % 0.36

 (0.10; 0.60)
3.19*

(2.78; 3.65)
8.02*# 

(6.75; 8.73)
leukocytes with signs of necrosis, % 0.83 

(0.65; 0.98)
0.86

 (0.73; 0.98)
1.39*# 

(1.20; 1.61)

Notes: * – the difference between the control and the experimental group is statistically significant (р<0.05-0.001)
           # – the difference between the 2nd and the 3rd study groups is statistically significant (р<0.05)

fig. 1. Caspase-3 level in case of carrageenan intoxication (* – significant difference compared with the 
control group, # – significant difference compared with the experimental groups).

0

50

100

150

200

250

300

control group 2 group 3

* 

*# 

%
 

surface [13]. Caspase-3 is the most involved 
pathway which should be generated from its 
inactive protein (procaspase-3), caspase 3 is 
required for some apoptosis features (chromatin 
condensation, DNA damage and apoptotic body 
formation) and its part may take place before 
cell viability suppression starts [14]. Hridneva SV 
notes that in chronic enterocolitis endothelial 
functions are impaired, which manifests itself in 
the activation of free radical oxidation processes 
with underlying decrease in the activity of the 
antioxidant system that explains the excessive 
production of ROS [15] and cell death. The 
activity of caspase-3 increases with increased 
carrageenan concentration, that proves a more 
obvious enterocyte apoptosis with the increase 
in daily intake of carrageenan. At the same time, 
animals of the 3rd group have a more obvious 
increase in the activity of caspase-3 in serum, 
indicating the increase in the severity of apo-
ptotic processes in intestine. The obtained data 
contradicts the results of some studies that prove 
that exposure of human intestinal epithelial cells 
to carrageenan in vitro does not lead to the 
activation of caspase-3, caspase-7 or increased 
percentage of fragmented DNA, suggesting no 
apoptotic alterations following carrageenan 
exposure [16]. Otherwise, the results of other 

studies evidence that carrageenan induced 
chronic gastroenterocolitis is accompanied by 
the decrease in the activity of PARP and elevation 
of MMP-2, MMP-9 and caspase-3 in blood serum 
of animals [17]. The results are controversial and 
require detailed consideration. Therefore, further 
research is warranted to elucidate the role of 
carrageenan in intestinal caspase-depending 
cell death that may help define novel nutritional 
strategies for hindering the development of gut 
diseases.

Conclusions
Oral use of carrageenan in rats was accom-

panied by the increase of the number of leuko-
cytes with signs of apoptosis: V+/PI-- cells in the 
2nd group increased in 1.9 times, and in the 3rd 
group – in 2.2 times, V+/PI+-cells increased in 
8.9 and 22.3 times, compared with the control 
(p<0.001); the percentage of leukocytes with 
later signs of apoptosis was significantly 
increased too (p<0.001). Animals that consumed 
1.0 % solution of carrageenan had more 
obvious increase in the activity of caspase-3 in 
serum relative to the group of rats consuming 
0.5 % carrageenan, proving the increase in the 
severity of apoptotic processes in intestine with 
the increase in the dose of carrageenan.



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ko  ТО, gorbach tV, Korobchanskyy Vo. Method of 
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Received: 2018-05-11

O.M. Kopanytsia et al.

КАРАГІНАН СПРИЧИНЯЄ ЗАГИБЕЛЬ КЛІТИН КРОВІ ЩУРІВ

О. М. Копаниця, М. І. Марущак, І. Я. Криницька
ТЕРНОПІЛЬСЬКИЙ ДЕРЖАВНИЙ МЕДИЧНИЙ УНІВЕРСИТЕТ ІМЕНІ І. Я. ГОРБАЧЕВСЬКОГО, ТЕРНОПІЛЬ, УКРАЇНА

Вступ. Завдяки своїм фізико-хімічним властивостям карагінан досить широко застосовується, а близько 70% 
усього виробленого в світі продукту використовується в харчовій промисловості. Попередні дослідження вказують 
на те, що введення карагінану при моделюванні хронічного ентероколіту викликає розвиток оксидативного стресу. 

Метою дослідження стало вивчення рівня апоптозу та некрозу в суспензії лейкоцитів у щурів при застосуванні 
0,5% і 1,0% розчинів карагенану.

Методи. Аналіз зв’язування анексину V (V) проводили з використанням набору Sigma Aldrich, США; рівень каспази 
у суспензії лейкоцитів та лімфоцитів крові щурів визначали спектрофотометрично.

Результати. Встановлено, що при застосуванні карагінану відсоток лейкоцитів з ознаками апоптозу в обох 
експериментальних групах статистично достовірно збільшувався. Активність каспази-3 зростала в 1,5 рази у 2-й 
групі і в 2,8 рази в 3-й групі у порівнянні з контрольними даними, які вказують на каспазо-залежний шлях апоптозу 
клітин при введенні карагінанy.

Висновки. Пероральне застосування карагінану в щурів супроводжувалося збільшенням кількості лейкоцитів 
з ознаками апоптозу. Тварини, які споживали 1,0% розчину карагінану, мали більш значуще збільшення активності 
каспази-3 в сироватці відповідно до групи щурів, які споживали 0,5% каррагінану, що доводить дозо-залежний ефект 
апоптотичних процесів у кишечнику.

КЛЮЧОВІ СЛОВА: карагінан; апоптоз; каспаза-3; щури.