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European Journal of Pharmacology 551 (2006) 156 – 161
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Montmorillonite ameliorates hyperthyroidism of rats and mice
attributed to its adsorptive effect
Yan Cai a , Xin-fang Meng b , Yong-xiao Cao a,⁎, Hua Lu a , Shao-fei Zhu a , Liang-zhen Zhou a
a
Department of Pharmacology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi, 710061, PR China
b
Shaanxi Provincial Institute for Drug Conntrol, Xi'an, Shaanxi, 710061, PR China
Received 16 May 2006; received in revised form 19 August 2006; accepted 23 August 2006
Available online 7 September 2006
Abstract
The present study aims to evaluate the adsorbing effect of montmorillonite on thyroid hormone in the entero-hepatic circulation. The
concentration of thyroid hormone in the serum of hyperthyroidism model rats and in solution was measured by radioimmunoassay and ultraviolet
spectrometry, respectively. The body weight, temperature, and consumption of food and water were observed in hyperthyroidism model rats.
Furthermore, hypoxia tolerance, sodium-pentobarbital-induced sleep time, spontaneous activities were measured on hyperthyroidism model mice
after being treated with montmorillonite. Results showed that montmorillonite adsorbed thyroxin (T4) and triiodothyronine (T3) in vitro.
Montmorillonite at dosage of 1.0 g/kg and 0.3 g/kg decreased thyroid hormone levels on hyperthyroidism model rats; Montmorillonite (2.0 g/kg
and 0.6 g/kg) prolonged the sleep time, improved the hypoxia tolerant capacity and reduced the spontaneous activities of the hyperthyroidism
model mice. These results suggest montmorillonite has anti-hyperthyroidism effect attributed to its adsorptive effect.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Hyperthyroidism; Montmorillonite; Thyroid hormone; Entero-hepatic circulation
1. Introduction
Entero-hepatic circulation of thyroid hormone involves
transfer of hormone to intestines in bile from the liver and
reabsorption of some intestinal hormone, via portal blood back
to liver (DiStefano et al., 1992). The rate of entero-hepatic
circulation of thyroid hormone is about 30% in normal rats.
Thyrotoxic states are characterized by an increased enterohepatic circulation of thyroid hormones, as well as an increased
urinary and faecal excretion of both conjugated and free thyroid
hormones (Bergman et al., 1996; Nguyen et al., 1993). We
suppose that the level of thyroid hormone should be lower when
adsorptive substances are used to sequester thyroid hormone of
entero-hepatic circulation in intestine (Mercado et al., 1996).
Montmorillonite is a layered silicate with the property of
adsorbing organic substances either on its external surfaces or
⁎ Corresponding author. No. 76, Yanta West Road, Xi'an, Shaanxi Province
710061, PR China. Tel.: +86 29 8265 5140; fax: +86 29 8265 7764.
E-mail addresses: [email protected], [email protected] (Y. Cao).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.08.053
within its interlaminar space, by the interaction or substitution
of the exchange cations presented in these spaces (Ramos and
Hernández, 1996). It is well known that smectite produced in
France is a popular drug for children's diarrhea. And now
montmorillonite is widely used as feedstuff additive and drug
carrier (Lin et al., 2002). Also it is found to be a catalyzer of
polymerization of amino acid adenylates because of its
adsorption on amino acid. However, to date, no study has
been conducted to test its effect for hyperthyroidism. The study
was thus conducted to evaluate its adsorption effectiveness on
thyroid hormone in vitro and in vivo.
2. Materials and methods
2.1. Reagents
Montmorillonite was provided by Hainan Derun Motmorillontite Factory. Thyroxin (T4) and triiodothyronine (T3) were
purchased from Sigma Chemical Co., St. Louis. Sodium
levothyroxin was from Shenzhen Zuolian Pharmacy Co. Ltd.
Y. Cai et al. / European Journal of Pharmacology 551 (2006) 156–161
Thyroid tablets was bought from Yanzhou Shengbao Pharmacy
Co. Ltd. Assay kits for total T3 and total T4 were from Chinese
Atomic Energy Academy of Science.
2.2. Animals
ICR mice weighing 18–22 g and male Sprague–Dawley rats
weighing 180–220 g were purchased from Animal Center of
Xi'an Jiaotong University School of Medicine. All animals were
handled according to the guidelines provided by the Animal Care
and Use Committee at Shaanxi Province. The animals were
housed in groups, maintained at 21–24 °C on a 12-h light and
12-h dark cycle, with free access to water and standard rat food.
2.3. Adsorption experiments in vitro
One percent of sodium dodecyl sulphate (SDS) stock solution containing T3 or T4 (50 μg/ml) was prepared, and was
diluted to the solutions containing 10 μg/ml. Scan was made by
ultraviolet–visible spectrophotometer from 200 nm to 400 nm
with 1% SDS as the control solution. A maximal absorption
wavelength of 230 nm was obtained.
Working solutions containing 40, 30, 20, 10, 8, 6, 4, 2 μg/ml
T3 or T4 were prepared with 1% SDS. Their absorption was
measured in 230 nm, and the standard curves were obtained. T3:
A = 0.0617C − 0.0022 (r = 0.9998); T4: A = 0.0691C + 0.030
(r = 0.9997).
T3 and T4 (10 μg/ml) were put into two 100 ml flasks,
respectively. Montmorillonite 300 mg was added into each flask
which was put in the 37 °C bath and shaken frequently.
Suspension (5 ml) was taken out from each flask after 5, 15, 30,
60, and 120 min, respectively. The suspension was filtered and
the absorption of the filtrate was measured (Lin et al., 2002;
Ramos and Hernández, 1996).
The T3 and T4 stock solutions were diluted to 50 μg/ml,
10 μg/ml and 2 μg/ml with 1% SDS, each of which was added
into a 10 ml flask containing 30 mg montmorillonite. A control
treatment without adsorbent was also carried out for each concentration. The flasks were put in a 37 °C bath and shaken
frequently. The adsorption of the filtrate was measured after 1 h.
T3 and T4 solutions (10 μg/ml) were put into 10 ml flasks
containing montmorillonite 300 mg, 30 mg and 3 mg, respectively. The adsorption was measured as above.
All the concentrations were calculated from the standard
curve, and adsorption rate (percentage) was calculated from the
following formula:
Adsorption rateðpercentageÞ ¼
C0 −C1
100%
C0
C0 is the concentration of T4 and T3 before montmorillonite, C1
is the remainder concentration after being adsorbed.
2.4. Hyperthyroidism model of rats
2.4.1. Anti-hyperthyroidism effect at different time
Rats were orally administrated with thyroid tablets 90 mg/
kg/day for 3 days, the serum concentration of total T3 and total
157
T4 was measured, Then the animals were divided into two
groups according to total T3 and total T4 level: model group and
montmorillonite group. Thyroid tablets of 90 mg/kg was given
to rats in the early morning daily. Montmorillonite of 300 mg/kg
was orally administrated 3 h after taking thyroid tablets in
montmorillonite group. Model group was given equivalent
volume of distilled water. Rat blood was taken by cutting their
tails at the 1st, 2nd, 3rd, 5th, 7th day after giving montmorillonite, and serum was separated by centrifuge, and total T3 and
total T4 level was determined.
2.4.2. Anti-hyperthyroidism effect at different doses
Rats were divided into six groups, each of which contained 10
rats and different treatments were given as following: normal
control group (distilled water); model group (thyroid tablets);
tapazol group (thyroid tablets + tapazol 15 mg/kg); montmorillonite 1.0 g/kg group (thyroid tablets + montmorillonite 1.0 g/kg);
montmorillonite 0.3 g/kg group (thyroid tablets + montmorillonite
0.3 g/kg); montmorillonite 0.1 g/kg group (thyroid tablets +
montmorillonite 0.1 g/kg). 90 mg/kg of thyroid tablets was orally
administrated once daily for 7 days. Montmorillonite or tapazol
was orally administrated 3 h after thyroid tablets (90 mg/kg/day
for 7 days, i.g.). The body weight, anorectal temperature, and
food and water consumption of the rats were measured after the
last drug was taken. Then rats' blood was taken from cutting
tails, the serum concentration of total T3 and total T4 was
measured.
2.4.3. Measurement of total T3 and total T4 in serum
The concentration of total T3 and total T4 was determined
with a highly sensitive, specific radioimmunoassay by a FM2000γ-counter. Radioimmunoassay kits of total T3 and total T4
were used according to the manufacture's instructions (Chinese
Atomic Energy Academy of Science, Beijing, China). The
serum samples and a series of standard T3 and T4 solutions were
added to tubes in which 125I–T3 or 125 I–T4 was subsequently
added. Then they were mixed with the anti-T3 (T4) microsphere
suspension and kept in 37 °C for 15 min. All the tubes were
centrifuged for 20 min afterwards, then the radioactive counts of
their supernatant were recorded. Therefore the standard curves
were obtained and the concentration of total T3 and total T4 in
serum could be calculated.
2.5. Hyperthyroidism model of mice
Mice were divided into 6 groups as: control; hyperthyroidism
model; tapazol 15 mg/kg; montmorillonite 2.0 g/kg; montmorillonite 0.6 g/kg; montmorillonite 0.2 g/kg. The mice received
sodium-levothyroxin (30 μg/kg/day) intraperitoneally for 7 days
except the control mice. At the same time, montmorillonite or
tapazol was orally administrated. On the 8th day, the mice were
put into the YLS-2A multifunctional spontaneous activity counter
and spontaneous activity was recorded. The sleep time of the mice
was recorded after being injected sodium-phentobarbital
(35 mg/kg, i.p.). The hypoxia-resisting time was recorded on
the mice, each of which was put in a 250 ml jar containing 15 g
of sodium lime and were airproofed with vaseline.
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Y. Cai et al. / European Journal of Pharmacology 551 (2006) 156–161
Table 2
The adsorbing effect of different concentration of montmorillonite on T4 and T3
(10 μg/ml)
Conc. of
T4
T3
montmorillonite
C0 − C1 Ca
Adsorption C0 − C1 Ca
Adsorption
(mg/ml)
(μg/ml) (mg/g) percentage (μg/ml) (mg/g) percentage
(%)
(%)
0.3
3.0
30.0
1.62
5.38
6.60
5.4
1.8
0.022
16.2
53.8
66.0
4.26
5.64
6.90
14
42.6
1.9
56.4
0.023 69.0
C0 is the concentration of T4 and T3 before adding montmorillonite, C1 is the
remainder concentration of T4 and T3 after being adsorbed. Ca is the amount of
T4 and T3 adsorbed per unit of weight of montmorillonite.
Fig. 1. The adsorption rate–time curves of montmorillonite on thyroid hormone.
Montmorillonite 300 mg was added to 100 ml solution of 10 μg/ml
triiodothyronine (T3) and thyroxin (T4). The suspension which contained
montmorillonite and T3 (or T4) solution was filtrated and the level of T3 and T4
in filtrate was measured at 5, 15, 30, 60, 120 min.
of T4 were 2, 10, and 50 μg/ml, the concentration of T4 adsorbed
by montmorillonite were 1.85, 5.25 and 7.74 μg/ml, respectively;
and adsorption percentage were 92.5%, 52.5%, and 15.5%,
respectively. Table 2 showed the adsorbing effect of different
concentration of montmorillonite on 10 μg/ml T4 and T3. When
the concentration of montmorillonite were 0.3, 3.0, and 30.0 mg/
2.6. Statistical analysis
The data are expressed as mean ± S.E.M. The one-way analysis of variance (ANOVA) followed by Dunnett's test was
applied for comparisons for more than two groups. And unpaired t-test was used when two sets of data were compared. A
two-tailed test with a P value less than 0.05 was considered
significant.
3. Results
3.1. Adsorptive effect of montmorillonite in vitro
The adsorption rate–time curve showed that montmorillonite
adsorbed thyroid hormone rapidly. The adsorption reached equilibrium in 30 min (Fig. 1). The maximal adsorption percentage of
T3 and T4 were 58.6% and 52.1%, respectively. Table 1 showed
the adsorbing effect of montmorillonite (0.3 mg/ml) on T3 and
T4. When the concentration of T3 were 2, 10, and 50 μg/ml, the
concentration of T3 adsorbed by montmorillonite were 1.76, 5.80
and 7.16 μg/ml, respectively; and adsorption percentage were
88.0%, 58.0%, and 14.3%, respectively. When the concentration
Table 1
The adsorbing effect of montmorillonite(0.3 mg/ml) on triiodothyronine (T3)
and thyroxin (T4)
Concentration (μg/ml)
T3
T4
C0
C1
C0 − C1
2
10
50
2
10
50
0.24
4.20
43.84
0.15
4.75
42.26
1.76
5.80
7.16
1.85
5.25
7.74
Adsorption
percentage (%)
88.0
58.0
14.3
92.5
52.5
15.5
C0 is the concentration of T4 and T3 before montmorillonite, C1 is the remainder
concentration after being adsorbed.
C −C
Adsorption percentage ¼ 0 1 100%.
C0
Fig. 2. The effect of montmorillonite on concentration–time curve of total T3
(Fig. 2A) and total T4 (Fig. 2B). Data represents the mean ± S.E.M for 10 rats.
⁎, ⁎⁎ indicate significant differences from model group, P < 0.05 and P < 0.01,
respectively. In the first 3 days the thyroid hormone level was increased as the
times of giving thyroid tablets increased. After being administrated with
montmorillonite the concentration of total T3 and total T4 were significantly
lower than the model group after the 5th day.
Y. Cai et al. / European Journal of Pharmacology 551 (2006) 156–161
159
Fig. 4. Effect of montmorillonite on food and water consumption of
hyperthyroidism model rats prepared by administrating thyroid tablets
(90 mg/kg/day, i.g.) for 7 days. The data represents the mean ± S.E.M for 10
rats. ⁎ indicates significant difference from model group, P < 0.05.
food consumption of the rats at the dose of 1.0 g/kg was
significantly less than the model group. The body weight and
anorectal temperature of montmorillonite group rats had a
decreased trend, but there was no statistical difference compared
with model group (data not shown).
3.3. Effect of montmorillonite on hyperthyroidism model mice
Fig. 3. The effect of montmorillonite on the serum concentration of total T3
(Fig. 3.A) and total T4 (Fig. 3B) in hyperthyroidism model rats prepared by
administrating thyroid tablets (90 mg /kg, i.g.) for 7 days. The data represents the
mean ± S.E.M. for 10 rats. ⁎, ⁎⁎ indicate significant difference from model
group, P < 0.05 and P < 0.01 respectively.
Fig. 5 showed that montmorillonite decreased spontaneous
activity and prolonged sodium-pentobarbital-induced sleep time
of hyperthyroidism model mice induced by sodium-levothyroxin
30 μg/kg intraperitoneally for 7 days. The count values of spontaneous activity became lower with montmorillonite 2.0 g/kg and
0.6 g/kg. Montmorillonite at 2.0 g/kg, 0.6 g/kg and 0.2 g/kg
groups prolonged the sleep time of model group (P < 0.05) which
was shorter than normal control group (P < 0.01).
Fig. 6 showed the effect of montmorillonite on hypoxia
tolerance capacity of hyperthyroidism model mice induced by
ml, the adsorption percentage of montmorillonite on T4 were
16.2%, 53.8% and 66.0%, respectively; and the adsorption
percentage on T3 were 42.6%, 56.4% and 69.0%, respectively.
3.2. Effect of montmorillonite on hyperthyroidism model rats
The concentration–time curves of total T3 and total T4 in
serum showed that levels of total T3 and total T4 in serum were
increased after thyroid tablets were administrated. The levels of
total T3 and total T4 in montmorillonite group became lower
from the fifth day on rats administrated with montmorillonite
than the model rats (P < 0.05 and P < 0.01). (Fig. 2)
The effect of montmorillonite on the serum concentration of
thyroid hormone in hyperthyroidism model rats showed that
montmorillonite decreased both total T3 and total T4 levels
obviously (Fig. 3). Montmorillonite (1.0 g/kg and 0.3 g/kg)
decreased the total T3 and total T4 levels of model group which
was higher than normal group.
Fig. 4 showed that effect of montmorillonite on food and
water consumption of hyperthyroidism model rats. The water
consumption of the rats in the 1.0 g/kg and 0.3 g/kg montmorillonite group were less than the model group (P < 0.05), and
Fig. 5. Effect of montmorillonite on spontaneous activity and asleep time
induced by sodium-pentobarbital in hyperthyroidism model mice induced by
sodium-levothyroxin (30 μg/kg/day, i.p.) for 7 days. Montmorillonite decreased
spontaneous activity and sleep time compared to the model group. Data
represents the mean ± S.E.M, for 10 mice. ⁎, ⁎⁎ indicate significant difference
from model group, P < 0.05 and P < 0.01, respectively.
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Y. Cai et al. / European Journal of Pharmacology 551 (2006) 156–161
Fig. 6. Effect of montmorillonite on hypoxia tolerance capacity in hyperthyroidism model mice induced by sodium-levothyroxin (30 μg/kg/day, i.p.) for
7 days. Montmorillonite (2.0 g/kg and 0.6 g/kg) improved hypoxia tolerance
capacity compared to the model group. ⁎ indicates significant difference from
model group, P < 0.05.
sodium-levothyroxin 30 μg/kg intraperitoneally for 7 days.
Montmorillonite (2.0 g/kg and 0.6 g/kg) prolonged the hypoxia
tolerance time compared to the model group (P < 0.05).
4. Discussion
The patients with thyrotoxicosis often show symptoms
resulting from excess circulation thyroid hormone (Solomon
et al., 1993). Thyroid hormone is conjugated with glucunronides and sulphates in liver. The conjugated products are excreted in the bile and entered the intestine, where bacteria can
degrade conjugated hormone to free T3 and T4. Free hormones
are reabsorbed in intestine. An enterohepatic circulation forms
as a result (DiStefano et al., 1993). About 1/3 of thyroid hormone participates in the enterohepatic circulation. The ratio is
obviously increased in hyperthyroidism. It has been suggested
that serum total T3 and total T4 level can decrease if thyroid
hormone entering intestine from enterohaptic circulation is
sequestered by sorbents, which may be beneficial to hyperthyroidism (Shakir et al., 1993; Surks and Sievert, 1995).
The study in vitro showed that montmorillonite could adsorb
thyroid hormone. In the in vivo experiment, the level of serum
total T3 and total T4 in hyperthyroidism model group was much
higher than the control group. In addition, the consumption of
water and food, body weight and anorectal temperature in
hyperthyroidism model group was increased compared to the
control group, demonstrating that the model was successful.
Similarly, the model mice which were injected with levothyrixin
became analepsia, and their oxygen consumption increased, too.
Montmorillonite significantly decreased total T3 and total T4
levels and water consumption of hyperthyroidism model rats.
Montmorillonite also decreased spontaneous activity and prolonged hypoxia tolerance time and sodium-pentobarbital-induced
sleep time of hyperthyroidism model mice. These suggest
montmorillonite has an effect on anti-hyperthyroidism, which
was attributed to its adsorptive effect. Montmorillonite not
only adsorbs free T3 and T4, blocking the enterohepatic
circulation of thyroid hormone, but also adsorbs intestine
bacteria which is necessary for the degradation of conjugated
hormone to free T3 and T4, decreasing free T3 and T4 level in
intestine. What's more, montmorillonite has the ability to
cover the surface of the intestine and prevents the reabsorption
of the hormone. These may involve in the anti-hyperthyroidism effect of montmorillonite.
Montmorillonite doesn't enter the body circulation, so its side
effect will be much slighter compared to other anti-hyperthyroidism drugs such as tapazol and propylthiouracil which would
induce granulocyte reduction and immunity deficiency (Qiu,
2002). Therefore, it is lucky for patients who are subtly to have
leukocyte reduction after taking the medicine of tapazol and
propylthiouracil. Also, it may be used as an adjunctive therapy,
that is, to combine with tapazol or other drugs.
Cholestyramine, an ionic exchange resin, can interfere with
the absorption of ingested thyroid hormone, too. It has showed
that combination therapy with propylthiouracil, propranolol and
cholestyramine in the treatment of Graves' hyperthyroidism
achieves a euthyroid state more rapidly and completely than that
with only methinmazole and propranolol (Tsai et al., 2005). Its
anti-hyperthyroidism effect was also attributed to its adsorptive
effect. 50 mg of cholestyramine can bind approximately 3 mg of
T4 (Harmon and Seifert, 1991), which blocks reabsorption of
thyroid hormone in enterohaptic circulation. Constipation and
abdominal discomfort are the primary side effects of holestyramine treatment. Heart burn, nausea and transient increase
in serum aminotransferase level occur, too.
Honestly, the adsorptive effect of montmorillonite is also
physical, not special. So other active substances or nutritions in
intestine may be adsorbed if it is used for a long time (Kozak
and Domka, 2004). So, it is necessary to study some important
substances like vitamins and iron and so on, to find out if
montmorillonite adsorbs them and how much the adsorption
will be.
Acknowledgements
We would like to thank Dr. Shemin Lu for his critical reading
and linguistic correction of the manuscript.
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