Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

EVALUATION OF ACUTE AND SUB-CHRONIC ORAL TOXICITY OF ETHANOLIC

EXTRACT OF CARDIOSPERMUM HALICACABUM IN MOUSE MODEL
Uvarajan Sampath*, Gopinath Gopal,Gayathri Margandeyan
Department of Biochemistry, Indo-American College, Cheyyar, Tamilnadu, India.
ARTICLE INFO
Article history
Received 08/10/2014
Available online
31/12/2014

Keywords
Necropsy,
MCV, MCH,
MCHCS,
Cardiospermum Halicacabum
Ethanolic Extract(CHEE).

ABSTRACT
Cardiospermum halicacabum Linn. (Sapindaceae) is a climber, commonly found throughout
India, used as a vegetable as well as traditional medicine for rheumatism and nervous
diseases. The purpose of this study is to evaluate the acute and sub-chronic oral toxicities of
an ethanol extract of Cardiospermum halicacabum. An acute oral toxicity study conducted by
giving dosage of 50, 100, 500, 1000, 2000 mg/kg bw of CH extract and the animals noted
after 1 h administration of the extract and examined after 14 days of administration for
mortality and morbidity. In sub-chronic oral toxicity studies, the selected oral doses 100,200,
500, mg/kg bw and the evaluation carried out after 90 days of oral administration of
Cardiospermum halicacabum extract Biochemical, and hematological changes with the body
and relative organ weights of the mice analyzed. The results of acute toxicity study
statistically analyzed using one way ANOVA which suggested there is a significant change in
body weight, organ weight function of liver and kidney and hematological parameters above
500 mg/kg bw. These findings point out the ethanolic extract of Cardiospermum halicacabum
owns toxic effect on the dosage above 500 mg/kg of bw and the LD 50 found to 900 mg/kg bw.
In subchronic oral toxicity study the biochemical and haematological parameters of
Cardiospermum halicacabum ethanolic extract treated mice have not shown any significant
changes.

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Please cite this article in press as Uvarajan Sampath et al. Evaluation of Acute and Sub-Chronic Oral Toxicity of Ethanolic
Extract of Cardiospermum Halicacabum in Mouse Model. Indo American Journal of Pharm Research.2014:4(12).

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Corresponding author
Uvarajan Sampath
Department of Biochemistry,
Indo-American College, Cheyyar, Tamilnadu, India
uvasambath@gmail.com

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INTRODUCTION
Cardiospermum halicacabum (Linn), family Sapindaceae, is a deciduous, branching, herbaceous climber, which spread
throughout the plains of India. The whole plant is use to treat rheumatism, stiffness of limbs, snakebite its roots used to cure nervous
diseases, as a diaphoretic, diuretic, emetic, laxative, refrigerant, stomachic sudorific ; its leaves and stalks use to treat diarrhea,
dysentery and headache as a poultice for swellings [1]. Phytochemical constituents such as flavones, aglycones, triterpenoids,
glycosides and fatty acids and volatile ester have reported from the various extracts of this plant [2]. There are no advisable data about
acute and subchronic oral toxicity of CHEE (Cardiospermum halicacabum ethanol extract) in mouse model. The present study
undertaken to evaluate the comprehensive acute and subchronic toxicity in the mouse model of Cardiospermum halicacabum.
MATERIALS AND METHODS
Extraction
The leaves dried at room temperature, crushed to powder and stored in a plastic container. For extraction, 150 g of air dried
powder extracted with ethanol (40-60oC) in a Soxhlet extractor for 18-20 h and the solution evaporated to dryness under reduced
pressure and controlled temperature by using a roto evaporator. The yields of the prepared extract around 12% w/w and the same
stored at -20oC.
Experimental Animals
Toxicity studies conducted using Swiss Albino Mice of both sexes. Swiss Albino Mice aged 6-7 weeks, weighing 2025 g
each used. The animals kept under standard conditions of humidity, temperature and light (12 h dark and 12 h light). The animals fed
with standard diet and water ad libitum and used after an acclimatization period of at least 10 days in the laboratory environment. The
animals deprived of food for 24 h before experimentation. The protocol approved by the Institutional Animal Ethics Committee of
Indo-American College, Cheyyar. (IAEC NO; /M02/13).
Chemicals
All chemicals used for the study purchased from Qualigens, India.
Acute Toxicity test
Acute toxicity test performed according to organize Economic Co-operation and Development (OCED) guideline 423. Mice
divided into groups of ten each (5 males and 5 females). CHEE dissolved in 10% Tween 20 and gave orally by gavage feeding (50,
100, 500, 1000, 2000 mg/kg bw). After administration of extract, mice noted for 24 h, with special attention given to the first 4 h and
once daily further for 14 days. The mice weighed and visual observations of mortality, behavioral pattern (salivation, fur, drowsiness,
and sleep), changes in physical appearance, injury, pain, and signs of illness conducted once daily during the period. At the end of the
experiment, Blood samples collected via cardiac puncture into nonheparinized and EDTA-containing tubes for biochemical and
hematological analyses, respectively. After cardiac puncture, the mice euthanized through intraperitoneal injection of a cocktail
containing ketamine (80 mg/kg bw) and xylazine (10 mg/kg bw). The organs excised, weighed, and examined microscopically.
Hematological and biochemical analyses performed. LD50 studied by Miller and Tainter (1944) method.
Sub-Chronic oral toxicity
Sub chronic oral toxicity test performed according to OECD Guidelines 408. The animals divided into three groups of ten in
each (5 males and 5 females). The doses chosen are 50,100, and 200 mg/kg bw based on 1500 3000 mg/ 60 kg bw of human beings
which may be 10 - 30 times for sub-chronic toxicity studies. Group 1 served as control received 10 ml/Kg of hot distilled water.
Groups 2, 3, and 4 given 200, 500, and 1000 mg/Kg bw of CHEE respectively for 90 Days daily at the same time and noted for
morbidity and mortality for two times daily. Mice kept on an ad libitum diet and tape water throughout the study. Body weights of the
animals evaluated weekly.

Statistical Analysis:
All the data earned expressed as mean SD. Differences in means estimated by ANOVA followed by Dunnets after this
test. The results considered significant at p<0.05.

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Hematological Assay
Tubes with EDTA as an anticoagulant used to collect whole blood for to study Hemoglobin, RBC, WBC, platelet, MCV,
MCH, and MCHC. All test methods adopted from Varley, Gewenlock, and Bell 1991.

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Effect of CHEE on liver role

At the end of 90 days of CHEE administration, blood collected after an overnight fasting without anticoagulant from the mice
in a plain tube which centrifuged at 3,500 rpm for 5 min. After Centrifugation, serum collected for estimating glucose (Method of
Marks 1959 using O-Tolidine), urea (DAM-TSC Method as described by Nateson et al., 1951), creatinine (JAFFs Method), alkaline
phosphatase, aspartate amino transferase (AST) (Modified Rietman Frang Method), alanine amino transferase (ALT) (Modified
Rietman Frang Method), bilirubin ( Malloy and Evelyn, 1937).

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RESULTS
Acute Toxicity:
In acute oral toxicity studies, found that the CHEE treated animals safe up to a maximum dose 500 mg/kg bw. The animals
not presented any abnormal signs and symptoms on cage side observatory procedures. In a gross necropsy study it viewed there no
significant change in the external surface of the body, orifices, cranial, thoracic and abdominal cavities. Body weights recorded. LD 50
identified to 900 mg/kg bw.

Fig 1. Acute toxicity result expressed as mean body weight of mice after 2 weeks treatment with CHEE shown significant
changes in 1000 mg/kg bw and 2000 mg/kg bw mice ( *p<0.05).
All values expressed as meanSD; n=10
3.5

*
*1

organ weight

2.5

Control
50 mg/kg

100 mg/kg
1.5

500 mg/kg

1000 mg/kg
*

0.5

2000 mg/kg

Liver

Kidney

Heart

Lungs Pancreas

Testis

Fig 2. Acute toxicity result expressed as mean organ weight of mice after 2 weeks treatment with CHEE shown significant
changes in liver, kidney and pancreas compared with control mice (*p<0.05).

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All values expressed as meanSD; n=10

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Table 1. Haematological parameters for mice after 14 days treatment with CHEE at different doses.
Parameter
Control
50 mg/kg
100 mg/kg
500 mg/kg
1000 mg/kg
2000 mg/kg
Hemoglobin g/dl
11.281.68
10.271.46
11.322.10
10.641.48
8.81.32*
8.81.26*
RBC (X 106/l)
7.361.21
7.541.32
7.421.62
6.681.36
5.421.24*
4.221.16*
WBC (X 103/l)
7.561.46
7.341.34
7.261.36
7.421.83
5.321.16*
3.42.98*
Platelet (X 103/l) 840.6270.54 810.5648.62 812.4954.26 720.1528.32 640.3626.52* 580.7236.46*
MCV(fl)
28.364.52
30.865.48
32.643.96
24.873.16
22.424.86*
18.672.46*
MCH(pg)
12.722.68
12.462.92
12.622.84
8.421.18
7.621.14*
5.461.26*
MCHC (%)
26.525.56
28.475.28
26.484.72
20.823.46
18.562.48*
15.461.17*
Haematocrit (%)
42.166.88
44.766.82
45.428.12
40.744.96
32.875.64*
22.463.92*
Result expressed as meanSD; (n=10) CHEE treated group showed significant changes in 1000 mg/kg bw and 2000 mg/kg bw
compared with control mice ( *p<0.05). All values expressed as meanSD; n=10.
Table 2. Biochemical parameters for mice after 14 days treatment with CHEE at different doses.
Parameter
Control
50 mg/kg
100 mg/kg
500 mg/kg
1000 mg/kg
2000 mg/kg
Glucose mgdL-1 81.468.92
81.426.46
61.325.48
61.124.36
58.323.98*
56.324.16*
Urea mgdL-1
128.4618.32 126.2714.56 124.3216.34 126.3212.84 116.5814.46 106.3212.34
CR mgdL-1
1.60.4
1.40.2
1.40.4
1.20.3
0.740.08
0.260.04
ALKP (U/L)
64.1616.32
62.3214.97
62.4815.68
58.4814.32
50.8612.46
50.2412.34
AST (U/L)
260.3220.46 262.4518.86 250.3214.48 258.5418.62 213.3416.64 180.8216.82
ALT (U/L)
54.3412.68
52.4614.36
51.3612.84
38.328.96
32.468.84
24.324.62
Bilirubin mgdL-1 0.370.08
0.260.04
0.320.03
0.280.06
0.120.02
0.120.03
Result expressed as meanSD; (n=10) CHEE treated group showed significant changes in 1000 mg/kg bw and 2000 mg/kg bw
compared with control mice (p<0.05). All values expressed as meanSD; n=10.
Sub-Chronic oral toxicity:
In sub-chronic oral toxicity studies the doses chosen (50,100,200 mg/Kg bw) found that no toxicity signs and symptoms in
any treated group. At the end of the treatment, the visceral condition of all the animals in the treated groups found normal compared to
the control groups.

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Fig 3. Sub Chronic oral toxicity result expressed as mean organ weight of mice after 90 days treatment with CHEE shown
significant changes in liver, kidney and pancreas compared with control mice (*p<0.05).All values expressed as meanSD; n=10.

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Table 3. Haematological parameters of mice treated with CHEE at different doses.

Parameter
Control
50 mg/kg-1 bw 100 mg/kg-1 bw 200 mg/kg-1 bw
Hemoglobin g/dl
10.261.18
10.531.12
10.701.17
10.551.25
RBC (X 106/l)
7.311.12
7.511.16
7.511.28
7.351.23
WBC (X 103/l)
8.331.80
8.551.6
8.451.2
8.631.4
Platelet (X 103/l) 773.0040.35 754.1647.73
770.1640.20
761.1643.40
MCV(fl)
32.585.2
32.554.8
32.835.8
32.454.6
MCH(pg)
13.411.2
14.711.6
16.631.3
15.381.4
MCHC (%)
24.514.11
24.455.12
24.484.14
24.634.19
Haematocrit (%)
37.415.11
38.715.17
38.316.14
37.485.4
Result expressed as meanSD; (n=10) CHEE treated group showed no significant changes compared with control mice ( *p<0.05). All
values expressed as meanSD;n=10.
Table 4. Biochemical parameters of mice treated with CHEE at different doses.

DISCUSSION
For the pharmacological evaluation of medicinal plant to assess the toxic characteristics of a natural product extract, fraction,
or compound are usually [3]. Determination of LD 50 is usually an in the assessment and evaluation of the toxic characteristics of a
substance. It is an early assessment of toxic manifestations and is one of the early screening experiments performed with all
compounds. Data from the acute toxicity study may; (a) Serve as the basis for classification and labeling ; (b) Provide first information
on the of toxic action of a substance; (c) Help arrive at a dose of a new compound; (d) Help in dose determination in animal studies;
(e) Help control LD50 values provide many indices of potential types of drug [4]. In acute toxicity test the significant changes in the
relative organ weights and body weight noticed only in the dose above 500 mg/kg bw which may suggest the extract might have toxic
potential on the organs like liver, kidney and pancreas. According to Moore and Dalley an increase in organ body weight ratio is a
suggestion of inflammation while a decline in the same parameter can be deducted to cellular constriction [5]. In this study, no
significant differences noted in the animals treated (P<0.05) with the dosage of 500 mg/kg bw and less compared to control (fig 1 &
fig 2). Therefore, this study suggests CHEE does cause acute toxicity effects above 500 mg/kg bw at the various dose tested and the
LD50 identified to be 900 mg/kg bw.
In subchronic toxicity test administration of CHEE for 90 days produced no clinical signs for toxicity or mortality in either
sex in the dosage of subchronic(fig 3). In addition, the CHEE treated mice did not show any significant change in water or food intake
(data not shown). Decline in food and water intake is suggested as being responsible for the decrement in body weight gain. Loss of
appetite is often Significant synonymous with weight loss due to disturbances in carbohydrate, protein or fat metabolisms [6]. Besides,
at higher doses, crude plant extracts may metabolise to a toxic product, which interfere with gastric role and decreased food
conversion efficiency [7]. Interestingly, the food and water intakes found to be unaltered during the 90 th day treatment period when
compared to a control group. Assessment of hematological features can be used to control the extent of harmful effect of extracts on
the blood of an animal. It can also be used to explain blood relating roles of a plant extract or its products [8]. Such analysis is relevant
to risk evaluation as changes in the haematological system have higher predictive value for human toxicity, when the data are
translated from animal studies [9]. Hemogram estimated for all the treated as well as control groups and results have shown no
unusual effect on hemoglobin, RBC, WBC, Platelet, MCV, MCHC, and haematocrit compared to control group (Table 3). MCHC,
MCH and MCV individual red blood cells while Hb, RBC and PCV are associated with the total population of red blood cells.
Therefore, the absence of significant effect of the extract on RBC, Hb, MCH, MCHC and MCV mean neither to incorporate
hemoglobin into red blood cells or the morphology and osmotic fragility of the red blood cells altered [10].
Expressing toxicity of xenobiotics is usually determined biochemically by recording of some plasma enzymes and lipids.
Normally, ALT, AST, AKP is found inside liver cells. But if the liver is inflamed or injured, ALT, AST, AKP are released into the
bloodstream. A rise in AST, ALT, ALP and bilirubin are commonly measured as indices of the damage of the liver cells [11]. Thus,
the changes in activity and concentration of tumour marker enzymes like AST, ALT and ALP in tissue such as liver reflect the state of
hepatotoxicity [12]. Our results revealed no significant changes (P<0.05) in the liver enzymes of the treated groups when compared
with the control. The values of glucose, urea, creatinine, ALP, AST, ALT and bilurubin close to the control group thus CHEE does not

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200 mg/kg bw
83.6612.67
125.3314.6
1.20.55
57.256.33
268.018.55
53.2510.35
0.300.70
compared with control mice (p<0.05).All

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Parameter
Control
50 mg/kg bw 100 mg/kg bw
Glucose mgdL-1 85.6512.38 87.6511.12
86.4510.35
Urea mgdL-1
130.214.6
128.215.36
122.2514.64
CR mgdL-1
1.10.3
1.20.45
1.50.62
ALP (U/L)
59.165.40
60.556.25
61.836.54
AST (U/L)
288.6319.4 290.3313.66 280.3611.25
ALT (U/L)
55.2613.4
59.9112.55
58.2511.65
Bilirubin mgdL-1 0.280.06
0.300.50
0.310.65
Result expressed as meanSD; (n=10) CHEE treated group showed no significant changes
values expressed as meanSD;n=10.

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alter the kindney and Liver function(Table 4). This suggests sub chronic administration of Ethanolic extract of cardiospermum
halicacabum leaves has no hepatotoxic effects in mice.
In conclusion, the acute oral toxicity of the ethanol extract of Cardiospermum halicacabum is safe up to 500 mg/kg bw, mild
significant changes were noticed at the level of 1000 mg/kg bw significant changes were encountered at the level of 2000 mg/kg bw.
Further the LD50 was identified as 900 mg/kg bw. In Subchronic oral toxicity studies the animal were safe at the selected different
dosage level .Hence the level of 500 mg/kg bw is safe for future pharmacological appliances.
Competing Interests
The authors declare no conflict of interest.
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