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Evaluation of acute oral toxicity of Butterfly Pea Root extract on experimental

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Conference Paper · November 2013

DOI: 10.1109/ICICI-BME.2013.6698516

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2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
Bandung, November 7-8, 2013

EVALUATION OF ACUTE ORAL TOXICITY OF

BUTTERFLY PEA ROOT EXTRACT ON
EXPERIMENTAL MICE
Jessica Karta1, Dr. rer. nat. Maruli Pandjaitan2,
and Dr. Min Rahminiwati3
1
Department of Biomedical Engineering, Swiss German University, Serpong, Indonesia
(Tel : +6281-21-8777-37; E-mail: karta.jessica@gmail.com)
2
Department of Biomedical Engineering, Swiss German University, Serpong, Indonesia
(Tel : +62-21-3045-0045; E-mail: maruli.pandjaitan@sgu.ac.id)
3
Departement of Anatomy, Physiology and Pharmacology, Bogor Agricultural University, Bogor, Indonesia
(Tel : +62-251-8629462; E-mail: minrahminiwati@ipb.ac.id)

Abstract- Clitoria ternatea (CT), is an herbaceous
medicinal plant used to treat brain related disorders and
dementia. The purpose of this study was to test the acute oral
toxicity of the extracts of roots plant. Acute toxicity of Butterfly
Pea Roots extract was evaluated in DDY-mice. The acute toxicity
studies were adopted from OECD guidelines 423 and Lu (1995).
The animals were orally administered a single dose of 2500, 5000,
10000 and 15000 mg/kg body weight. Signs of toxicity and
mortality were noted after 1, 4 and 24h of administration of the
extract for 14 days. The highest dose administered (15000mg/kg
I. INTRODUCTION
Over the years, the study of plants as alternative
medicine, or more commonly known as herbal remedies, has
grown enormously around the world. There is a lot of
evidence that shows medicinal plants have great potential for
curing various diseases and medical disorders [31].
The extract from the roots of Butterfly Pea, or
commonly known as Clitoria ternatea, has been used in
traditional Indian medicine as a brain tonic due to its
perceived ability to promote memory and intelligence. A study
revealed that the root extracts could increase rat brain acetyl
choline content, a vital neurotransmitter in both peripheral
nervous system (PNS) and central nervous system (CNS). The
root inhibits the activity of enzyme acetylcholinesterase,
which degrades the amount of acetylcholine in a similar
fashion to the standard cerebro drug Pyritinol, known as an
anti-Alzheimer’s drug according to some researchers [35].
Indonesian people have long believed in the miracles
of traditional herbal medicine (jamu), and it has been used
since ancient times both for preventive or therapeutic
treatments. Indonesia is a tropical country with abundant and
overwhelming natural resources for herbal medicines which
means that Indonesian people have had more opportunities to
make discoveries than most other cultures. Clitoria Ternatea
body weight) produced the highest mortality rate and the LD50 of
Butterfly Pea roots extract is 32118.533 mg/kg based on Probit
Analysis. The histopathology studies indicate hepatotoxicity and
nephrotoxicity as the acute toxic effect of Butterfly Pea root
extract.
Keywords: Clitoria ternatea, Acute Toxicity, Dementia, LD50,
Histopathology.
flower, locally known as “Kembang Telang”, has been used
for centuries throughout generations to treat red eye, ulcers,
earache, fever, distended abdomen, irritation in the bladder
and urethra, as well as eliminating phlegm in chronic
bronchitis [5]. This fascinating plant can also be cultivated
easily in this country, either privately at home or for industrial
purposes.
A previous study strongly indicated that the extract of
Clitoria ternatea root could increase memory and learning
abilities of mice [32]. However, the toxicity test of the purified
root extract has not been performed yet. This research,
therefore, attempts to investigate the suspicious toxicity
effects of Clitoria ternatea root extract. It is crucially
important for new medicines or drugs to meet safe
pharmacology standards before being made available to the
public. The safety and efficacy of plants as alternative
medicine must also be established before they are legally
permitted to be used as therapeutic agents.
The scope of this research is the study of acute oral
toxicity which underlines the median lethal dose (LD50) of
Clitoria ternatea root extract. Microscopic examination
(histopathology test) is also performed on target organ toxicity
in order to better understand the unrecognized toxic effects on
mice as appropriate animal models. For all types of toxicity,
the cause and effect relationship between chemical exposure
and illness leads to the crucial information of dose-response


 


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2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
Bandung, November 7-8, 2013

relationship. It is believed that the right dose differentiates a
poison from a remedy [17]. It intention of this study try and
establish the overall acute toxicological effect of the Butterfly
Pea root extract.
II. MATERIALS AND METHODS
Collection and Extraction of Plant Material
food was withheld but not water). Experimental mice were
distributed into one control group and four treated groups of
different levels of dose of Clitoria ternatea root extract, with
four animals in each group. Levels of treatment are indicated
in Fig. 2 below in line with the animal groupings.
I Group II Group
Dosage : 2500 mg/kg bw Dosage: 5000 mg/kg bw

The roots from Butterfly Pea plants (age between 5 Control Group
months and 2 years old) were collected from 2 local areas of 1 cc Aquadest
private plantation in Cimanggu, Bogor, Jawa Barat and III Group
up IV Group
Pamulang, South Tangerang. The roots then were put through Dosage:10000 mg/kg bw Dosage: 15000 mg/kg bw
a series of extraction processes described in Fig. 1 as follow:
Butterfly Pea Root Fig. 2. Treatment Levels for Acute Toxicity Study of Butterfly Pea Root
Washing Extract.
Cutting Following period of fasting, treated mice were
Drying (45°C
(45°C, 24 hours) weighed and the amount of test substances delivered was
Grinding calculated. Doses were prepared shortly before being
Sieving (0
(0.63mm)
63mm)
administered to each treatment group in order to maintain the
test substances’ stability before it was administered to each
Addition of Solvent (1:10)
treatment group. Maximum dose administration volume was
Solvent Extraction (25°C
(25°C, 4 hours
hours, speed 7) 1ml/kg of mouse body weight. Once the dose of each
Centrifugation (25°C,
(25°C 8000rpm
8000rpm, 15 mins treatment group was administered, the food was withheld for
Supernatant 1-2 hours. The time of death was recorded and the number of
Freeze Drying
g ((-50°C
(-50°C,
50 48 hours) survivors was noted after 24 hours. The mortality rates within
24 hours were used to determine the median lethal dose
Final Product of (LD50) of Clitoria ternatea root extract.
Clitoria ternatea Root Extract For clinical data, treated animals were observed
individually soon after the dose of each treatment levels was
Fig. 1. Flowchart of Butterfly Pea Root Extraction Process.
successfully administered. Period of observation was the first
The percentage yield of dry extract Clitoria ternatea root was 30-mins, periodically during the first 24-h, with special
found to be 5.14% w/w. attention given during the first 4-h, and daily thereafter, for a
total of 14 days. Any symptoms of ill-health or abnormal
Experimental Animals behavior were taken into account. Parameters of toxic signs
were studied according to OECD-423 guideline, including
Male-DDY mice (± 20-35 gram BW) were obtained tremors, convulsions, salivation, diarrhoea, lethargy, sleep and
from the Animal Housing, Faculty of Medicine, University of coma. Skin and fur, eyes and mucous membrane, respiratory,
Indonesia, Salemba Raya, Central Jakarta. All mice were circulatory, autonomic and central nervous systems,
randomly divided into several groups and housed in plastic somatomotor activity and behavior pattern are the other
cages (40x25x10cm). The animals were kept under standard parameters which were also noted.
environmental laboratory conditions (temperature: 25±2°C). The study was maintained for a further 14 days with
Lighting was controlled of sequence 12-h light and 12-h dark daily observation on individual body weight and food
for each 24-h period. consumption. The amount of food delivered was controlled;
A 14 days period of acclimation was followed for the each mouse was given 10 grams of diet food per day.
mice to adapt to experimental lab-condition, prior to the
starting day of primary research. Standard diet (animal food Histopathology Examination
pellets) was delivered with water ad libitum, provided by the
Institute Pertanian Bogor, West Java, Indonesia. Animals found dead during the study were subjected to gross
necropsy. Five main organs including heart, lung, kidney, liver
Acute Toxicity Studies and brain were taken and rinsed with physiological saline
(0.9% NaCl) solution. Then, the targeted organs were
A single dose of test subtance (C.ternatea root preserved in 10% neutral buffered formalin and the samples
extract) was orally administered by gavage using stomach were all sent to BBalitvet, Bogor, West Java, where
tube. Treated animals were fasted 18-h prior to dosing (only

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2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
histopathology tests were conducted. At the end of the study,
the remaining mice that were still alive were humanely killed
for providing essential study information for this research.
Statistical Analysis
The LD50 of Clitoria ternatea root extract was analyzed by
linear regression probit analysis (Finney, 1971) using SPSS
computer program. The correlation of dosage vs gain in body
weight and dosage vs the amount of food intake for 14 days
from every treatment groups and a control group were
analyzed using two factors Analysis Of Variance (ANOVA)
without replication. The percentage of body weight gain was
calculated and other supporting data on food consumption of
each treatment group were evaluated through regression
statistics.
III. RESULTS AND DISCUSSIONS
Acute Toxicity and LD50 Determination
The investigation of acute oral toxicity of Clitoria
Ternatea or Butterfly Pea roots point out the median lethal
dose (LD50) of the roots extract is higher than 15000 mg/kg
bw. The highest mortality rate at 50% occurred at a dose of
15000 mg/kg bw. Common mortality rates at 25% were
obtained with the other dose levels range from 2500 to 10000
mg/kg bw.
Based on Probit analysis using SPSS software, the
LD50 of Butterfly Pea roots extract is 32118.533 mg/kg bw
with 95% confidence interval. Accordingly, Butterfly Pea
roots extract is classified as a practically non-toxic compound
(Lu, 2002). The value of LD50 obtained from this research is a
preliminary study resource for a higher toxicity evaluation that
requires more samples and should be conducted by a certified
toxicologist. Probit model for LD50 of Butterfly Pea root
extract is shown in table 1.
Clinical observation indicates the acute effects of
Butterfly Pea root extract impact the autonomic nervous
system (ANS). The ability of Clitoria ternatea (CT) roots
extract in promoting acetylcholine levels in
parasympathetic neurons may explain this pharmacological
action. It is strongly believed the adverse effect of CT roots
extract as a cholinergic agonist affecting both sympathetic and
parasympathetic division as high dose levels administered.
Too much acetylcholine might lead to a serious
medical condition resulting from its accumulation in the ANS
synapses. Physical signs of toxicity due to the acute exposure
of CT root extract ranged from decreased in locomotors,
depression, cyanosis, piloerection, sedation to relaxed
nictitating membrane which was followed by dyspnea,
convulsions and it led to death.
319
Bandung, November 7-8, 2013
Daily observation on body weight and food
consumption of all surviving animals from each treatment
levels draws a further discussion. Based on weekly evaluation
as shown in Fig. 3, the highest body weight gain is indicated
in group with dose level 10000 mg/kg bw and follow by dose
15000 mg/kg bw, 5000 mg/kg bw and 2500 mg/ kg bw
sequentially. The body weight of control group was apparently
decreasing.
ANOVA results show the dose levels influence the
body weight gain and food consumption of animals (p-value
<0.05) in every treatment group. These facts led to the
reversible toxic effect of Butterfly Pea root extract according
to Lu, 2002. Indeed, signs of toxicity seemed to be disappear
following the termination of standard period of observation.
During the clinical period, the surviving animals seemed to
recover in 2 hours after the delivery of doses.
The relation of body weight gain and food
consumption of treatment groups and the one control group
were evaluated. Based on regression analysis, there was no
correlation between the body weight gain and food intake at
dose levels 15000 mg/kg bw and 10000 mg/kg bw (p-value >
0.05). There is a correlation between gain in body weight and
food intake at lower treatment levels which are 2500 mg/kg
bw, 5000 mg/kg bw and the control group.
This study revealed the administration of Clitoria
ternatea roots extract significantly increases the appetite and
body weight of each treated mouse. The highest levels of
treatment at 15000 mg/kg bw of Clitoria Ternatea roots extract
led to the biggest appetite levels. This indicates that Clitoria
ternate roots extract may have the potential to increase
appetite and body weight. Further research focusing on the
effect of Butterfly Pea roots to body weight gained and food
consumption should be conducted. At present, histopathology
results of the toxic effects of CT roots extract provide essential
data to draw more accurate conclusions.
AVG Body Weight Gained (%)
30.00 26.26
25.00 21.21 20.08
17.19
20.00
13.30
15.00 11.45
10.00 6.65 6.00
5.00 -1.79 0.05
0.00
2500 5000 10000 15000 control
-5.00
Groups of Treatment
Day 7 Day 14
Fig. 3. Graphic of Percentage of Body Weight Gain of each
Treatment Group for 14 Days.
2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
Bandung, November 7-8, 2013

Table 1. Probit Model of LD50 Butterfly Pea Roots Extract.

Groups *Concentration Log conc. No. of animals % Probit LD50

(mg/kg bw) Exposed Dead Kill Kill (mg/kg bw)
I 2500 3.398 4 1 25 0.210
II 5000 3.699 4 1 25 0.279 32118.533
III 10000 4.000 4 1 25 0.356
IV 15000 4.176 4 2 50 0.405
V Control for Groups I-IV

Histopathological Studies

occur in either the liver or kidneys at this dose level. This

Levels of toxicity were indicated from the
strongly indicated that mice which had the highest dose
histopathology studies of 5 targeted organs of toxicity: liver,
administered were those that suffered the most severe damage
kidneys, brain, heart and lung. Histopathology results
on liver and kidneys. This fact leads to the conclusion that the
obtained from Bbalitvet were evaluated between the dead and
acute exposure of Butterfly Pea roots extract has the most
surviving animals.
destructive effect on organs liver and kidneys.
Histopathology results from the mice that died due to
Evaluation of the histopathology results and its
acute exposure of CT root extract at different dose levels
correlation to the amount of body weight gained and food
indicate the most severe damage occurred in the liver and
intake also allows us to draw further conclusions. Previously it
kidneys. Dead mice which received the highest amount of
was indicated that increasing amount of food intake and body
Butterfly Pea roots extract at dosage 15000 mg/kg bw were
weight gained corresponded to higher exposure of Butterfly
found to have tubular necrosis on the kidneys and hepatic
Pea roots extract at dosage 15000 mg/kg bw and 10000 mg/kg
necrosis. Vacuolization also occurred in the liver and
bw. In fact, histopathology results of surviving mice at these
infiltration mononuclear cell in the kidneys. The dead mouse
dose levels indicate severe damage occurred in its organs
with 10000 mg/kg bw was investigated and found to have
especially its livers and kidneys. Therefore, body weight gain
peripheral hepatic necrosis, polymorphonuclear and
and increasing amount of food intake is not a desirable
infiltration mononuclear cell in the liver. While in the kidneys,
condition in this case. Details on histopathology of the liver
tubular necrosis and infiltration of mononuclear cell had
and kidney from the control, death and surviving mice are
occurred.
given in the following Fig. 4, Fig. 5, Fig. 6, Fig 7, Fig. 8, Fig.
At 5000 mg/kg bw, midzonal hepatic degeneration,
9, Fig. 10 and Fig.11.
polymorphonuclear, congestion and infiltration of
Histopathology examination indicated more severe
mononuclear cell occurred in the liver. The mouse kidneys
damage to the liver and kidneys in the mice that died from
were investigated and found to have multifocal tubular
exposure compare to the surviving mice at higher dose levels
necrosis and hemorrhage. At the lowest dose level
of Clitoria Ternatea. Further studies on tissue damage need to
(2500mg/kg bw), the liver was diagnosed as having midzonal
be conducted focusing on the nervous system in order to have
hepatic degeneration, polymorphonuclear, vacuolization and
a more complete information of the toxic effects of Butterfly
infiltration of mononuclear cell. Note that necrosis did not
Pea roots extract.

320
2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
Bandung, November 7-8, 2013

3
2

1
2 1

Fig. 4. Liver (Control). Fig. 5. Liver (Survival). Dosage: 5000 mg/kg bw.

A normal liver treated as a control group. Hepatic cells (1). Portal tract (2). HE. Midzonal hepatic necrosis showing focal hepatic necrosis (1), portal tract (2),
20x. central vein (3) and midzonal hepatic degeneration (4). HE. 20x.

5 2

1
3
2 1

3
4

Fig. 6. Liver (Death 1). Dosage: 10.000 mg/kg bw. Fig. 7. Liver (Death 2). Dosage: 15.000 mg/kg bw.

A dead animal showing acute hepatic necrosis. Haemorrhage (1), necrosis of A dead animal showing hepatic degeneration. Hepatic cells degeneration (1),
hepatic cells (2), polymorphonuclear cells (3), vacuolisation of hepatic cells (4) dilatation of sinusoid (2) and hepatic cells vacuolisation (3). HE. 20x.
and infiltration of mononuclear cells (5). HE. 20x.

321
2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
Bandung, November 7-8, 2013

1
1

Fig. 8. Kidney (Control). Fig. 9. Kidney (Death 1). Dosage: 5000 mg/kg bw.

A normal kidney treated as a control group. Normal glomerulus (1) and normal A surviving animal showing tubular necrosis (1) and hemorrhage (2). HE. 20x.
tubular (2). HE. 20x.

Fig. 10. Kidney (Survival). Dosage: 10.000 mg/kg bw. Fig. 11. Kidney (Death 2). Dosage: 15.000 mg/kg bw.

A dead animal showing tubular necrosis (1). HE. 20x. A kidney showing tubular necrosis (1) and infiltration of mononuclear cells
(2). HE. 20x.

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2013 3rd International Conference on Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME)
IV. CONCLUSION
From the experiment it can be concluded the predicted LD50
of Butterfly Pea Roots Extract for DDY-Mice is 32118.533
mg/kg bw, which is the classification of a practically non-
toxic compound according to Lu, 2002. Clinical signs of
toxicity point out the adverse effect of Butterfly Pea Roots
Extract on the autonomic nervous system (ANS). Body weight
gain and food consumption indicate the reversible toxic signs
of Butterfly Pea Roots Extract. Histopathology results show
the acute exposure of Butterfly Pea Roots extract led to
relatively severe damage on liver and kidneys.
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