Tài liệu Evaluation of subchronic toxicity of tri thien duoc hard capsule in experimental animals – Nguyen Thi Thanh Loan: 10 JMR 116 E3 (7) - 2018
JOURNAL OF MEDICAL RESEARCH
EVALUATION OF SUBCHRONIC TOXICITY OF TRI THIEN DUOC
HARD CAPSULE IN EXPERIMENTAL ANIMALS
Nguyen Thi Thanh Loan1, Pham Thi Van Anh1,
Vu Thi Ngoc Thanh1, Nguyen Thi Ngoc Tram2
1Ha Noi Medical University
2Thien Duoc Limited Company
Tri Thien Duoc, a formulation of Portulaca oleracea L. and Amaranthus spinosus L., is aimed to treat
hemorrhoids and other related conditions; however, the safety of this product’s long-term consumption has
never been reported. The present study evaluated the subchronic toxicity of Tri Thien Duoc hard capsule in
experimental animals. Subchronic toxicity was studied in Wistar rats based on the guidance of World Health
Organization and Organisation for Economic Co-operation and Development. The rats were treated with the
doses of 0.566 g extract/kg per day and 1.698 g extract/kg per day for a period of 90 consecutive days. Tri
Thien Duoc hard capsule caused no significant dose-relat...
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10 JMR 116 E3 (7) - 2018
JOURNAL OF MEDICAL RESEARCH
EVALUATION OF SUBCHRONIC TOXICITY OF TRI THIEN DUOC
HARD CAPSULE IN EXPERIMENTAL ANIMALS
Nguyen Thi Thanh Loan1, Pham Thi Van Anh1,
Vu Thi Ngoc Thanh1, Nguyen Thi Ngoc Tram2
1Ha Noi Medical University
2Thien Duoc Limited Company
Tri Thien Duoc, a formulation of Portulaca oleracea L. and Amaranthus spinosus L., is aimed to treat
hemorrhoids and other related conditions; however, the safety of this product’s long-term consumption has
never been reported. The present study evaluated the subchronic toxicity of Tri Thien Duoc hard capsule in
experimental animals. Subchronic toxicity was studied in Wistar rats based on the guidance of World Health
Organization and Organisation for Economic Co-operation and Development. The rats were treated with the
doses of 0.566 g extract/kg per day and 1.698 g extract/kg per day for a period of 90 consecutive days. Tri
Thien Duoc hard capsule caused no significant dose-related changes in general status, hematological pa-
rameters, renal and hepatic function tests; in addition, it did not cause any change in histology of the liver
and kidney of the rats. Our finding obtained that the Tri Thien Duoc hard capsule did not cause subchronic
toxicity in experimental animals.
Keywords: Tri Thien Duoc, subchronic toxicity, experimental animals
I. INTRODUCTION
Hemorrhoids are a very common anorectal
disorder defined as the symptomatic enlarge-
ment and abnormally downward displacement
of anal cushions. They affect millions of
people around the world and represent a
major medical and socioeconomic problem [1;
2]. Hemorrhoids usually present with itching,
rectal pain, or rectal bleeding. Patients with
hemorrhoidal disease may experience any of
the following symptoms: bleeding, a painful
anal mass, swelling, discomfort, discharge,
hygiene problems, soiling and pruritus [3; 4].
According to the dentate line, hemorrhoids can
be divided into internal and external hemor-
rhoids. Some hemorrhoids are regarded as
Corresponding author: Nguyen Thi Thanh Loan,
Department of Pharmacology, Hanoi Medical University
Email: nguyenthanhloan@hmu.edu.vn
Received: 10/7/2018
Accepted: 15/11/2018
mixed hemorrhoids [5; 6]. Treatment of symp-
tomatic hemorrhoids ranges from dietary
advice, lifestyle modification and pharmacol-
ogical approaches to office-based procedures
and radical surgery depending on their grade
and severity [7]. Treatment of hemorrhoids in
modern medicine is still in its infancy. Due to
limited modern pharmacotherapeutic options
available for treatment, herbal medicines
remain the therapy of choice. The Tri Thien
Duoc hard capsule, a polyherbal proprietary
formulation, is aimed to treat hemorrhoids and
other related conditions. Tri Thien Duoc is for-
mulated from bioactive flavonoids extracted
from Portulaca oleracea L. and Amaranthus
spinosus L. To date, there are no systematic
scientific studies to delineate its toxic effects
on experimental animals. Therefore, the pre-
sent study was investigated to evaluate the
subchronic toxicity of the Tri Thien Duoc hard
capsule in experimental animals.
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JOURNAL OF MEDICAL RESEARCH
II. MATERIALS AND METHODS
1. Plant materials
The Tri Thien Duoc hard capsule consti-
tutes a mixture of extracts of Portulaca
oleracea L. and Amaranthus spinosus L. One
capsule contains two herbal materials corre-
sponding to 0.59 g extract. This capsule is
dissolved in water before oral administration.
The Tri Thien Duoc hard capsule is pro-
vided by Thien Duoc Co.,Ltd according to the
principles of Good Manufacturing Practice
(GMP), Good Laboratory Practice (GLP) and
Good Storage Practice (GSP).
2. Experimental animals
Normal healthy Wistar albino rats weighting
between 160 g and 200 g were obtained from
the animal center of Dan Phuong, Ha Noi. The
animals were allowed an acclimatization pe-
riod of 7 days to laboratory conditions prior to
the initiation of the study. They were main-
tained for 12 hour light and dark cycles in a
well-ventilated house, with free access to food
and water ad libitum. All animals were treated
according to international regulation on experi-
mental animal treatment.
3. Experimental design
A subchronic toxicity study was carried out
according to guidance of World Health Organi-
zation and Organisation for Economic Co-
operation and Development [8; 9].
A total of thirty Wistar albino rats were di-
vided into three groups of ten animals:
- Group 1 (control group): orally adminis-
tered 1 ml/100g per day sterile distilled water;
- Groups 2 (treated group): orally adminis-
tered Tri Thien Duoc at dose 0.566 g extract/
kg per day
- Groups 3 (treated group): orally adminis-
tered Tri Thien Duoc at dose 1.698 g extract/
kg per day
Tri Thien Duoc was orally administered
daily for a period of 90 consecutive days by
oral gavages.
Blood with EDTA was used immediately for
determination of hematological parameters
(total red blood cells, hematocrit, hemoglobin
concentration, total white blood cells and
platelet count). Standardized diagnostic kits of
Hospitex Diagnostics (Italy) and DIALAB
GmbH (Austria) were used for determination
of the following biochemical parameters:
alanine aminotransferase (ALT), aspartate
aminotransferase (AST), total bilirubin, albu-
min, total cholesterol and creatinine. All serum
biochemistry was performed using biochemi-
cal analyzer Erba Chem. Hematological analy-
sis was performed using automatic hemato-
logical analyzer Exigo - Boule Medical AB.
At the end of the experiment (after blood
collection), the kidney and liver were removed,
cleaned with saline solution and preserved in
10% formalin for histopathology examinations.
4. Statistical analysis
Data were analysed using Microsoft Excel
software (2007). The levels of significance
between the experimental groups and the con-
trol were made using the student's t-test. Data
are shown as mean ± standard deviation. All
data were considered significant at p < 0.05.
III. RESULTS
1. Effect on general status
No animal mortality was recorded in the
treatment groups throughout the study period.
No significant differences in the average body
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weights were observed between the treated
groups and the control group (p > 0.05). None
of the animals in any treated groups showed
any macroscopic or gross pathological
changes when compared to the control group.
2. Effect on hematological parameters
Table 1. Effect of Tri Thien Duoc on total red blood cells
Days
Total red blood cells (T/l) p
(t-test student) Group 1 Group 2 Group 3
Before 7.31 ± 0.76 7.10 ± 0.78 6.99 ± 0.43 > 0.05
After 30 days 7.57 ± 0.54 7.32 ± 0.62 7.32 ± 0.30 > 0.05
p (before-after) > 0.05 > 0.05 > 0.05
After 60 days 7.05 ± 0.78 6.86 ± 0.76 6.90 ± 0.48 > 0.05
p (before-after) > 0.05 > 0.05 > 0.05
After 90 days 6.94 ± 0.89 7.03 ± 0.80 7.09 ± 0.65 > 0.05
p (before-after) > 0.05 > 0,05 > 0.05
After 90 days of treatment, repeated daily oral administration of Tri Thien Duoc at oral doses of
0.566 g extract/kg/day and 1.698 g extract/kg/day did not cause significant changes (p > 0.05)
when comparing the treated groups to the control.
Figure 1. Effect of Tri Thien Duoc on
hemoglobin concentration Figure 2. Effect of Tri Thien Duoc on
hematocrit
As shown in figures 1 and 2, there is no significant difference in hematocrit, hemoglobin con-
centration between the treated groups and the control group (p > 0.05).
As summarized in table 2, white blood cell values of groups that were treated with Tri Thien
Duoc showed no difference when comparing the treated groups to the control group (p > 0.05).
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JOURNAL OF MEDICAL RESEARCH
Table 2. Effect of Tri Thien Duoc on total white blood cells
Days
Total white blood cells (G/l) p
(t-test student) Group 1 Group 2 Group 3
Before 7.39 ± 0.90 6.93 ± 1.31 7.35 ± 1.25 > 0.05
After 30 days 7.14 ± 1.52 7.44 ± 1.75 7.51 ± 1.42 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
After 60 days 7.70 ± 1.13 7.28 ± 1.90 7.66 ± 2.08 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
After 90 days 7.61 ± 1.15 7.72 ± 1.33 7.25 ± 0.93 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
Table 3. Effect of Tri Thien Duoc on platelet count
Days
Platelet count (G/l) p
(t-test student) Group 1 Group 2 Group 3
Before 529.10 ± 107.92 566.80 ± 82.17 520.60 ± 132.02 > 0.05
After 30 days 494.70 ± 113.60 511.90 ± 136.01 560.20 ± 126.55 > 0.05
p (before - after) > 0,05 > 0,05 > 0,05
After 60 days 506.30 ± 112.32 535.80 ± 102.31 503.30 ± 94.36 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
After 90 days 514.50 ± 108.88 495.00 ± 133.80 493.40 ± 113.51 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
As observed in table 3, no significant difference in the platelet count was observed between
groups that were treated with Tri Thien Duoc at dose 0.566 g extract/kg/day and 1.698 g extract/
kg/day with the control group (p > 0.05).
3. Effect on liver damage
As shown in figures 3 and 4, there was no significant difference in AST and ALT values
between the treated groups and the control group.
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JOURNAL OF MEDICAL RESEARCH
Figure 3. Effect of Tri Thien Duoc on
aspartate amino transferase
Figure 4. Effect of Tri Thien Duoc on
alanine aminotransferase
4. Effect on liver function
Table 4. Effect of Tri Thien Duoc on total bilirubin, albumin and total cholesterol
Parameters Days Group 1 (1) Group 2 (2) Group 3 (3)
Total bilirubin
(mmol/l)
Before (a) 13.23 ± 0.68 13.45 ± 0.71 13.38 ± 0.42
After 30 days (b) 13.33 ± 0.65 13.32 ± 0.83 13.41 ± 0.59
After 60 days (c) 13.35 ± 0.45 13.24 ± 0.79 13.34 ± 0.53
After 90 days (d) 13.47 ± 0.56 13.56 ± 0.48 13.39 ± 0.50
p2-1 > 0.05, p3-1 > 0.05, pb-a > 0.05, pc-a > 0.05, pd-a > 0.05
Albumin (g/
dL)
Before (a) 3.99 ± 0.23 3.78 ± 0.26 3.82 ± 0.28
After 30 days (b) 3.84 ± 0.34 3.69 ± 0.29 3.76 ± 0.32
After 60 days (c) 3.65 ± 0.34 3.81 ± 0.29 3.69 ± 0.32
After 90 days (d) 3.74 ± 0.18 3.71 ± 0.34 3.81 ± 0.28
p2-1 > 0.05, p3-1 > 0.05, pb-a > 0.05, pc-a > 0.05, pd-a > 0.05
Total choles-
terol (mmol/l)
Before (a) 1.33 ± 0.24 1.20 ± 0.16 1.30 ± 0.23
After 30 days (b) 1.18 ± 0.23 1.27 ± 0.15 1.27 ± 0.37
After 60 days (c) 1.27 ± 0.16 1.16 ± 0.14 1.37 ± 0.33
After 90 days (d) 1.19 ± 0.17 1.25 ± 0.20 1.33 ± 0.26
p2-1 > 0.05, p3-1 > 0.05, pb-a > 0.05, pc-a > 0.05, pd-a > 0.05
As shown in table 4, serum levels of total bilirubin, albumin and total cholesterol of the treated
groups using Tri Thien Duoc at dose 0.566 g extract/kg/day and 1.698 g extract/kg/day were not
statistically different when compared to the control group (p > 0.05).
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JOURNAL OF MEDICAL RESEARCH
5. Effect on kidney function
The effect of subchronic oral administration of Tri Thien Duoc on the creatinine of the control
and treated groups is shown in the table 5. Repeated daily oral administration of Tri Thien Duoc
at oral doses of 0.566 g extract/kg/day and 1.698 g extract/kg/day did not cause significant
changes (p>0.05) when comparing the treated groups to the control.
Table 5. Effect of Tri Thien Duoc on creatinine
Days
Creatinine (mg/dl) p
(t-test student) Group 1 Group 2 Group 3
Before 1.06 ± 0.08 1.06 ± 0.07 1.06 ± 0.07 > 0.05
After 30 days 1.07 ± 0.07 1.05 ± 0.08 1.05 ± 0.10 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
After 60 days 1.06 ± 0.10 1.05 ± 0.08 1.06 ± 0.07 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
After 90 days 1.05 ± 0.07 1.05 ± 0.05 1.06 ± 0.11 > 0.05
p (before - after) > 0.05 > 0.05 > 0.05
6. Histopathological examination
Histopathological examination of the control group and the Tri Thien Duoc treated rats with
0.566 g extract/kg/day and 1.698 g extract/kg/day showed normal structure and absence of any
gross pathological lesions in the liver and kidney.
Group 1. Control group
(HE x 400)
Normal structure
Group 2. Treated group, Tri
Thien Duoc at dose 0.566 g
extract/kg/day
Normal structure
Group 3. Treated group, Tri
Thien Duoc at dose 1.698 g
extract/kg/day
Normal structure
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JOURNAL OF MEDICAL RESEARCH
Group 1. Control group
Normal structure
Group 2. Treated group,
Tri Thien Duoc at dose
0.566 g extract/kg/day
Normal structure
Group 3. Treated group,
Tri Thien Duoc at dose
1.698 g extract/kg/day
Normal structure
IV. DISCUSSION
Currently, the use of herbal medicines in
the treatment of hemorhhoids has expanded
rapidly in both developed and developing
countries. Data concerning toxicity of plants
are important as a baseline before exploring
the therapeutic potential of a new herbal medi-
cation. A subchronic toxicity study provides
information on the effects of repeated oral ex-
posure and can indicate the need for further
longer term studies [9; 10].
Body weight changes serve as a sensitive
indication of the general health status of
animals [10]. Weight gains were observed in
all animals administered with Tri Thien Duoc.
In addition, none of the animals in any treated
groups showed any macroscopic or gross
pathological changes when compared to the
control group. It can be stated that Tri Thien
Duoc did not interfere with the normal
metabolism of animals as corroborated by the
non-significant difference from animals in the
control group.
The hematopoietic system is one of the
most sensitive targets of toxic compounds and
is an important index of the physiological and
pathological status in animals [8; 9]. After 90
days of treatment, there was no significant
difference in total red blood cells, hematocrit,
hemoglobin concentration, total white blood
cells and platelet count between groups that
were treated with Tri Thien Duoc and the con-
trol group; therefore, it can be concluded that
the administration of Tri Thien Duoc did not
affect the hematological profile and blood for-
mation process. Similarly, Bhande Satish re-
ported the extract of Amaranthus spinosus did
not lead to any deleterious effects on hemato-
logical parameters in the animals treated at
125 and 250 mg/kg per day. The absence of
significant changes may suggest that Tri Thien
Duoc does not have toxic effects at these
dose regimens in albino rats [11].
The liver plays a key role in many meta-
bolic process of not only itself but of other tis-
sues as well. Severe hepatic injury, as a result
of the metabolism of some of the toxic phyto-
chemicals found in medicinal plants and failure
of the metabolic products to be eliminated by
the liver may be associated with marked
distortion of these functions. Total bilirubin,
albumin and total cholesterol are useful indi-
JMR 116 E3 (7) - 2018 17
JOURNAL OF MEDICAL RESEARCH
ces of the excretory function of the liver. Be-
sides, ALT and AST are useful indices for
identifying inflammation and necrosis of the
liver. Accordingly, the liver releases AST, ALT
and an elevation in plasma concentration is an
indicator of liver damage [9]. In view of the
serum biochemical parameters of the animals
treated with Tri Thien Duoc, the non-significant
changes in ALT, AST, total bilirubin, albumin
and total cholesterol in both male and female
rats at all doses indicates that Tri Thien Duoc
had no deleterious effect on liver function and
liver damage. Furthermore, histopathological
examination of the liver of the control group
and all treated groups did not reveal any mor-
phological differences. This is consistent with
the levels of AST, ALT and creatinine of
groups that treated Tri Thien Duoc were not
significantly different to the control group.
Kidney function analysis is very important
in the toxicity evaluation of drugs and plant
extracts. The biochemical analyses were done
to evaluate the possible alterations in renal
functions influenced by the plant products [12].
Concentration of creatinine can be used in
describing the function of the kidneys [9].
Creatinine level presented no significant differ-
ences between the control group and the
treated groups. In addition, histological studies
could present more information regarding the
nephrotoxicity of Tri Thien Duoc. The results
of this study indicate that histopathological
examination of Tri Thien Duoc treated rats
showed normal structure and absence of any
gross pathological lesion in the kidney; there-
fore, Tri Thien Duoc did not affect the kidney
function.
Overall, the findings of this study indicate
that there are no significant differences in
blood profiles or biochemical parameters. This
finding was further confirmed by histopa-
thological observations of the kidney tissue in
this study. In another study, we evaluated the
acute toxicity of Tri Thien Duoc hard capsules
in experimental animals. The results demon-
strated that the maximum tolerable dose of Tri
Thien Duoc is 7.9 g extract/kg/day, which is
five times higher than the administered dose in
this study. It caused no animal mortality in the
treated groups throughout the 90 day follow-
up. These observations indicate that Tri Thien
Duoc at the dose level of 0.566 g extract/kg
and 1.698 g extract/kg/day can be used for
further pharmacological activity.
V. CONCLUSION
The present study demonstrated that the
Tri Thien Duoc hard capsule at doses 0.566 g
extract/kg/day and 1.698 g extract/kg/day did
not produce any toxic signs or evident symp-
toms at subchronic oral toxicity.
ACKNOWLEDGMENTS
The authors thank the Professor Le Dinh
Roanh, director of the Center for Research
and Early Detection of Cancer, for reading
histology of liver and kidney of experimental
rats.
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