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Full Research Paper Influence of age on subchronic toxicity of the aqueous extract of the
leaves of Calotropis procera on rabbits
Article April 2011

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Journal of Toxicology and Environmental Health Sciences Vol. 3(5) pp. 119-126, May 2011
Available online at http://www.academicjournals.org/JTEHS
ISSN 2006-9820 2011 Academic Journals

Full Length Research Paper

Influence of age on sub-chronic toxicity of the aqueous

extract of the leaves of Calotropis procera on rabbits
Pouokam Guy B.1*, Hatem Ahmed2, Christianna Dawurung3, Aliyu Atiku3
Shamaki David4 and Okewole Philipe4
1

Laboratory of Food Sciences and Metabolisms, Department of Biochemistry, University of Yaound 1,Cameroon.
2
Chemical Laboratory, Medico-Legal Department, Ministry of Justice, Egypt.
3
Toxicology Unit, Department of Biochemistry, National Veterinary Research Institute, Nigeria.
4
Center Diagnosis: National Veterinary Research Institute, Nigeria.
Accepted 15 April, 2011

Calotropis procera is of the family Asclepiadaceae. The whole plant is toxic. The influence of age on
sub-chronic toxicity of aqueous leaves extract was investigated in New Zealand White X local breed
rabbits (Oryctolagus cuniculus) of both sexes. 200 mg/kg body weight of the extract was daily
administered orally by gavages during 42 days. Vital signs, body weight change, mortality, serum
biochemistry, hematology parameters, necropsy and histopathology were examined. All the rabbits
gained weight during the administration period, with an appreciable gain for smaller animals.
Significant decrease (p<0.05) of ALT and RBC were noticed in the youngest rabbits. A significant
increase (p<0.05) of serum creatinine level and lymphocytes were also noticed within the group of the
juvenile rabbits. Necropsy revealed lesions in kidney and liver, these lesions were further confirmed by
histopathology observations that revealed more pronounced pathology with the youngest animals.
Although animals in different test groups show some toxic effects; small animals of eight weeks exhibit
more effects with more severe lesions.
Key words: Age, Asclepiadaceae, histopathology, toxicity.
INTRODUCTION
Calotropis procera plant commonly called Sodom apple
or Giant milkweed belong to the family of
Asclepiadaceae. It is a major grazing plant found in Asian
temperate region (Arabian - Peninsula), Asia - Tropical
(India and Indo-China) and Africa, especially in semi arid regions. C. procera is abundant in the northern part
of Nigeria (Agaie et al., 2007).Preliminary phytochemical
screening of fresh leaves of C. procera in water extract
revealed the presence of phenols, saponins, tannins,
glycosides and mixtures of cardenolides (Murti et al.,
2010; Soto et al., 2011).
Total ash value of the powdered leaf of the plant has
been found to be 18.3% and water soluble ash 1.9%. As
many others plant of the African pharmacopeia C. procera

*Corresponding author. E-mail:pouokam_guy@yahoo.fr. Tel:

(+237) 77046789.

plant, plant extracts and pure isolated compounds from

natural sources provide a foundation for modern
pharmaceuticals. The medicinal potential of Sodom apple
has been known to traditional systems of medicine for a
while, with it leaves been widely used. Different parts of
the plant are used to treat diarrheoan stomatic, sinus
fistula and skin diseases. The leaf part is used to treat
jaundice (Murti et al., 2010). The whole plant (roots, bark,
stem latex and leaves) is toxic. Incidental ingestion of
fresh leaves of Giant milkweed has been suggested as
toxic to ruminants by several farmers from Brazilian semiarid regions. Administration of Giant milkweed leaves to
sheep as responsible of tachycardia and transitory
cardiac arrhythmia at auscultation 30 min after dosage.
C. procera plant is cardiotoxic and hepatotoxic poisonous
plant (Soto et al., 2011).
Aqueous and ethanolic roots and leaves extract of the
plant provoqued modification of vaginal smears with
perturbation of oestrus cycle in rats; characterized by

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J. Toxicol. Environ. Health Sci.

prolong disoestrus stage with temporary inhibition of

ovulation (Circosta et al., 2001).
Aqueous leaves extract administered orally to rabbits at
doses up to 80 mg/kg bw/day showed slight variations,
not statistically significant of AST, ALT, ALP and total
protein. In addition, fluctuation in values of hematological
parameters (PVC, RBC, platelets, and differential
leucocytes counts) was analyzed also to have no
statistical difference compare to control (Jato et al.,
2009). It is well known that metabolism of xenobiotics
varies with many factors amongst which the age and sex.
Sub-chronic toxicity effects of aqueous leaves extract
were found to exert minimal effects with no influence of
sex at dosage 80 mg/kg bw/day (Oyebandi et al., 2007
unpublished). In 2006, the Center Diagnosis laboratory of
the National Veterinary Research Institute (N.V.R.I) received
local farmers complaints pointing out dead of small animals
in their herd. A common point to all these complaints were
the presence of Calotropis plant around their grazing area;
therefore, in 2008, one of the aim of the annual Toxicology
Research Internship organize by the Africa Education
Initiative (NEF) foundation was to investigate influence of
age on the toxicity of C. procera as life stage is a
modulating factor of the toxicity (A. Atiku, National
Veterinary Research Institute, Nigeria, personal
communication). Present study aims at investigating the
influence of age on the sub-chronic toxicity of the
aqueous extract of the leaves of C. procera plant on
rabbits.
MATERIALS AND METHODS
Animals
Twenty-five New Zealand White x local breeds rabbits (Oryctologus
cuniculus) from 8 to 18 weeks old, of both sexes (0.55 to 2.30 kg
bodyweight) used during the study were obtained from the Small
Animal Unit, National Veterinary Research Institute, Vom, Nigeria.
Animals housed per group were acclimatized for 9 days before the
commencement of the experiment; they were maintained under
standard temperature and humidity conditions. They were daily fed
ad libitum with pelleted feed obtained from Dagwom Farms,
N.V.R.I, Vom. Each animal received subcutaneously and 10 days
before the administration period; antiparasitic drugs of large
spectrum against internal and external parasites at the dose of 1
mg/kg body weigh (Ivermectine). In addition, they received
anticocciciocidal (Toltrazuril), active against all intracellular
development stages of Eimeria spp, at dosage 1 ml/L for 2 days.
Rabbits were handled in accordance with the standard guide for the
care and use of experimental animals. All protocols followed were
validated and endorsed by the Institutional ethical committee of the
N.V.R.I.

were suspended in 1 L of water for 72 h, then filtrate to obtain the

stock solution.
237.85 g of dry powdered was obtained from 10 kg of fresh
leaves, given a percentage yield of 2.38% w/w with the extract
being dark brown in color and readily soluble in water.

Extract administration and duration

Extract was administered orally every morning between 8 to 9 am
by intubations for a period of 42 days, using a syringe fitted with a
catheter tube. All test animals were given 200 mg/ kg body weight
before their morning food, while control received equivalent volume
of distilled water.

Blood sample collection

7 to 10 ml of blood was collected from the ear of each animal using
hypodermal syringe before extract administration and before animal
sacrificed. One portion was immediately kept into a test tube
containing anticoagulant EDTA (Ethylene diamine tetra acetate) for
hematology analysis; it was centrifuged at 3000 rpm for 5 min using
a table centrifuge. The obtained sera were stored at - 4C prior to
analysis. The portion for biochemical analysis introduced into a
universal test tube and then store at - 4C.

Experimental design
Animals were allocated to five groups of five rabbits according to
their ages: Group A: 8 weeks; Group B: 10 weeks; Group C: 14
weeks; Group D; 18 weeks and a control group of 5 animals with
one animal being the control for each age group; except Group A
who had 2 controls. Blood samples were collected before extract
administration and before sacrificing the rabbits for biochemical and
haematological parameters analysis. Clinical signs were monitored
twice per day throughout the period of administration. At the end of
the experiment, all the surviving animals were sacrificed by
pumping air into their heart, they were necropsied and further
observations were done during histopathology.
Alanine amino transferases (ALT) and aspartate amino
transferases (AST) were determined as described by the Reithman
and Frankel (1957). Alkaline phosphatase (ALP) by the method of
King Armstrong (1964) modified from King and King Method (1954).
Serum creatinine level was determined by Roberry-Folin method,
while serum urea level was dosed by the diacetyl monoxime (DAM)
method.
Haematological analysis were perform: packed cell volume (PCV)
was analysed using the Micro-haematocrit machine, hemoglobin
(Hb) concentration was revealed by the cyanomethemoglobin
method; while red blood cell (RBC) was assess by Coles method
(1986). White blood cell (WBC) count was done using the improved
Nuebeauer counting chamber and differential leukocyte was by
microscopy of stained smears.

Statistical analysis
Plant collection and extract preparation
Fresh leaves of C. procera were collected from Fardan Karshe
village, Kaduna State, Nigeria. The plant was identified according to
the description by Felter and Lloyld (1898), and then authenticated
by comparing with the voucher specimens at the herbarium section
of the Federal College of Forestry, Jos, Plateau State, Nigeria.
Extraction was done (maceration) and 50 mg of the fine powder

Results were obtained after calculation with Excel software and

presented as mean standard deviation for all groups, using a
scatter gram, data were found to be normally distributed; student t
test was used to analyze differences (at p< 0.05) between pre and
post value for test and control animals. Analysis of variance
(ANOVA, 2 way) was done to check any significant difference (at
p<0.05) amongst various age groups. It should be noted that
because each control group had only one rabbits, the standard

Pouokam et al.

deviations results for control animals were expressed to be zero.

GRAPHPAD software was used for data analysis.

RESULTS
Bodyweight
All the animals was gaining weigth throught the
administration period with an appreciable gain for the
smaller rabbits (Group A, 8 weeks) 47.25%; almost the
same with control and test animals.
Clinicals signs observed
10 min after extract and distilled water administration,
rabbits showed an increase in heart bit (10 to 68%) and
respiratory rate ( 15 to 60%), with an important increase
for test group compare to the control. Moreover test
animals showed chewing movement of the mouth and
only those in group A hadskin hair loss at the end of the
administration period.
Biochemical and haematological analyses
Results of serum enzyme changes are shown in Table 1.
Significant decrease (at p <0.05) in values of ALT and
increase in creatinine were observed respectively for for
Groups A and B animals.
Table 2 shows the results of haematological analyses.
We find a significant increase (at p <0.05) of hemoglobin
in Groups A and B and an increase in PCV for Group B.
Differential leucocytes counts analysis (Table 3)
showed an increase of lymphocytes in Groups A and B
and and increase in monocytes for Group C animals.
DISCUSSION
Although C. procera has been reported to possess
various medicinal properties and toxics effects, no study
have been found reporting the impact of age on its
toxicity. This work investigates the influence of age on
sub-chronic toxicity of the aqueous leaves extract of the
plants on rabbits of different ages, ranging from 8 to 14
weeks.
Clinical signs observed were common to all animals in
test and control groups as reported by Jato et al. (2009);
except chewing movements of mouth observed only in
test animals and skin hair loss observed only in test
animals from Group A at the end of the administration
period. Increase in heart bit and respiratory rate could be
due to the animal stress due to handling during
administration, Bitterness of the extract due to the
presence of bitters active substance can explained
chewing of movements of rabbits mouths. No previous

121

skin hair loss were found in the literature after

administration of aqueous extract of the leaves of C.
procera to any others animals, this may be explained by
the strain of test animals, the dosage or the duration of
the administration period and also the age or life stage of
the animals. On the other hands, bodyweight is known to
be one of the most sensitive indicators of adverse effects.
All rabbits bodyweight was observed to increase (11.86
to 47.64%). Group A animals, however were gaining
weight more than others (47 to 64%). This weight gained
was also observed (Jato et al., 2009), but contrast with
another conclusion from Dada et al. (2002); reporting
weight loss during oral administration of latex of C.
procera in rats. This gained can be explained by the fact
that they are at stage of rapid growth and development.
Table 1 showed results of the effects of the aqueous
leaves extract of C. Procera on biochemical parameters;
before and after administration for tests and control
rabbits. Blood urea nitrogen (BUN) and serum creatinine
both estimates blood filtering capacity of the kidneys.
Variations in BUN and creatinine values for all groups
were not significant. Except a decrease in creatinine for
Group B. BUN has been shown to be significantly
elevated when the kidney function is reduced from 60 to
75% (Wallace, 2007); while an increase of creatinine was
observed in Group B, but was related to any other
increase for the same parameters in other group so as to
confirm a treatment effects. Creatinine is removed from
plasma by glomerullar filtration and excreted into urine.
Increase in creatinine values is an indication of renal
dysfunction (Smith and Hampton, 1990), this damage
could be due to the accumulation of active principles of
the plant extract into the kidney, accumulation of hazards
can be toxic to the tubular epithelial cells (Parke, 1982).
Although creatinine is more specific to determine kidney
injuries, our results could not confirm any harmful effects
to these organs by the extracts.
ALT and AST are cytoplasmic in location and get
releases in serum; an increase in the level of ALT, AST
and ALP is conventionally an indicator of liver injury (Chavda
et al., 2010). No significant change was noticed in AST and
ALP values, ALT values in smaller animals were found to
decrease significantly (at p <0.05), although the biological
significance of the ALT enzyme decrease is up till now
unclear, it is typically dismissed as being of no toxicology
importance. Potential causes reported to explain this
decreased includes: decreased hepatocellular production
or release of enzymes, inhibition or reduction of the
enzymes activity and interference with the enzyme assay
(ECETOC guidance document, 2002). Since liver is also
a major organ of protein synthesis, any decrease in liver
can be seen as damage of hepatocytes with alteration of
its production capacity or, in case of kidney damage and
increase loss of proteins or muscle wasting via
catabolism ( Wallace, 2007).
The decrease in hemoglobin concentrations of the
extract treated animals, further suggests that prolonged
administration of the extract may cause anaemia as

122

J. Toxicol. Environ. Health Sci.

Table 1. Effects of the aqueous leaves extract of C. Procera on Biochemical parameters; before and after (#) administration for tests and control rabbits.

Group A/ (8 wks)
Control
Tests
34, 0 0.0
44 22.56
() 52 0.0
52.5 26.6

Group B / (10 wks)

Control
Tests
53 0.0
59.4 59.1
61 0.0
51.25 18.87

ALT (IU)

31 0.0
() 45 0.0

37.7 12.84
8.7 12.29**

29 0.0
35 0.0

31.6 24.26
25.7 5.64

36 0.0
57 0.0

41.9 27.37
35.7 1.65

47 0.0
62 0.0

41.28 10.81
39.25 13.8

ALP (IU)

18.804 0.0
() 25.850.0

19.8825.30
28.56 9.99

8.2690.0
15.780.0

16.76 18.49
17.98 10.84

6.07 0.0
23.27 0.0

18.65 27.94
39.69 26.25

12.2250.0
33.130.0

13.42 10.51
44.96 42.77

Total protein (g/L)

92. 75 0.0
() 97.710.0

85.05 16.97
111.5 26.75

91.7890.0
100.660.0

84.77 20.13
106.32 11.39

96.94 0.0
73.76 0.0

103.5 39.51
126.62 37.8

91.0 0.0
120.040.0

104.95 43.16
99.72 13.23

Blood urea nitrogen IU)

3.56 0.0
() 3.770.0

4.48 1.48
3.06 1.13

4.00 0.0
8.60 0.0

3.92 0.925
2.48 0.57

4.22 0.0
6.910 0.0

3.64 1.31
2.11 0.321

1.67 0.0
9.3450.0

3.4 1.26
2.48 0.121

Creatinine mg/dl

0.312 0.0
() 2.770.0*

0.316 0.09
0.426 0.40

1.1 0.0
2.6 0.0

0.349 0.142
1.180.72*

0.190 0.0
1.91 0.0*

0.66 0.323
2.03 3.24

2.11 0.0
2.24 0.0

1.91 2.27
1.43 0.075

AST (IU)

Group C/ (14 wks)

Control
Tests
39 0.0
27.8 29.87
34.0 0.0
18.66 10.86

Group D/ (18 wks)

Control
Tests
9.0 0.0
3637.9
25 0.0
26.66 4.16

All values are expressed as mean SD, N=5; * significantly different from control (p< 0.05).

reported by Mahanmoud et al. (1979) and on the

contrary of Jato et al. (2009), due to the presence
of some toxic principles in the extract such as
saponins. It is a known fact that oral
administration of saponins containing compounds
could cause bloat, thereby reducing appetite in
animals (Trease and Evans, 1989). Eventhougth a
depression of RBC and Hg were seen, suggesting
anemic effects, only decrease in Hg was
significant, not RBC nor hematocrit. Moreover,
elevation in eosinophils cells number were found.
Eosinophils concentration increase is also
recognized as sign of pernicious anemia (Wallace,
2007), making the anemic effects not strongly
conclusive.
Table 3 shows results of differential leucocytes

counts. Statistical significant (at p <0.05) increase

of lymphocytes counts were found only for Groups
A and B, while a decrease in monocytes cells for
Group C (14 weeks) were also observed. An
elevation in white blood cells (WBC) is due to the
bone marrow stimulation, most often causes by
bacterial infections, while the exact causes of
monocytes depression is still unknown (Wallace,
2007).
Histopathological examination of liver and lung
sections of control Group A (Plates 1 and 3
respectively), show normal cellular architecture
with distinct hepatic cells, central vein and
sinusoidal spaces for liver and normal alveolar
structure for lung tissue. In the treatment group,
liver (Plate 2) exhibit perilobular toxic necrosis of

the portal area and lung (Plate 4) showed

thickened intraalveolar septae with edema fluid
and widespread hemorrhage. Observations of
liver and lung section tissues of control and
treated animals in Group A shows possible toxics
effects of the aqueous extract of the plants.
Although some of the lesions were common to all
the animals, the severity was mostly noticed for
the younger animals in the liver, lung.
Conclusion
The presents study demonstrates possible
influence of age on the toxic effects of aqueous
leaves of C. procera, administered repeatedly at

Pouokam et al.

123

Table 2. Haematological parameters before and after (#) administration for tests and control rabbits.

WBC10 /L
12

RBC 10 L
Hemoglobin (g/dl)
PCV (%)

Group A/ (8 wks)
Control
Tests
7.4 0.0
9.4 1.89
() 8.8 0.0
11.260.238
7.97 0.00
3.0570.74
() 7.90 0.0
3.730.291
16.3 0.0
13.751.98
() 10.3 0.0
10.130.815*
34 0.0
33.5 4.26
() 330.0
35.665.28

All values are expressed as Mean SD, N=5;

Group B / (10 wks)

Control
Tests
6.6 0.0
5.73 1.01
6.5 0.0
5.84 2.91
6.58 0.0
3.65 0.40
5.24 0.0
4.05 0.54
13.60.0
15.06 1.36
11.60.0
12.122.38*
380.0
31.331.33
370.0
37 5.33*

Group C/ (14 wks)

Control
Tests
5.8 0.0
3.025 1.28
6.0 0.0
6.225 3.6
6.72 0.0
3.45 0.863
5.16 0.0
3.52 0.815
17.30.0
12.85 2.79
10.20.0
11.52 2.54
41 0.0
31.254.93
35 0.0
36 5.59

Group D/ (18 wks)

Control
Tests
5.8 0.0
7.3 3.20
5.6 0.0
4.05 1.29
7.16 0.0
3.14 0. 83
6.69 0.0
3.53 0.48
14.60.0
13.27 1.35
10.80.0
12.98 10.3
38 0.0
31 6.55
390.0
37.51.29

Group C/ (14 wks)

Control
Tests
62 0.0
482.15
42 0.0
50.57.93
0.0 0.0
0.00.0
0.0 0.0
0.50.57
0.5 0.0
0.00.0
0.0 0.0
0.750.5
38 0.0
5012.31
57 0.0
496.68
0.0 0.0
2.00.52
1.0 0.0
0.750.5**

Group D/ (18 wks)

Control
Tests
48 0.0
531.41
54 0.0
44.510.59
0.0 0.0
0.00.0
0.0 0.0
0.50.75
0.00.0
0.00.0
0.00.0
0.750.095
50 0.0
462.82
460.0
53.511.70
2.0 0.0
1.01.41
0.00.0
0.750.5

* significantly different from control (p < 0.05).

Table 3. Differential leucocytes counts.

Neutrophils (%)
Eosinophils (%)
Basophils (%)
Lymphocytes (%)
Monocytes (%)

Group A/ (8 wks)
Control
Tests
59 0.0
528.48
()61 0.0
42.3310.60
0.0 0.00
0.00.0
()0.10.0
1.01.0
0.1 0.0
0.00.0
()1.20.0
0.330.55
56 0.0
3811.314
()760.00
75.6610.01**
1.0 0.00
1.02.82
()0.00.00
0.661.155

Group B / (10 wks)

Control
Tests
46 0.0
38.57.78
26 0.0
46.87.25
0.0 0.0
0.00.0
1.0 0.0
1.80.49
0.010.0
0.00.0
0.00.0
0.60.54
460.0
49.45.94
710.0
61.57.78*
4.0 0.0
0.00.0
1.0 0.0
1.40.54

All values are expressed as mean SD, N=5; * significantly different from control (p < 0.05).

administered repeatedly at dosage 200 mg/kg/bw

for 42 days; with most effects found in smallest
animals. It should be noted that findings in
biochemistry and hematological parameters were
not strongly conclusive because of the small
number of animals in control groups, choice of
age groups not large enough to significantly mark
without confusion a real age impact. In

conclusion, aqueous leaves extract of C. procera

administered during 42days at 200 mg/kg/bw in
rabbits from 8 to 14 weeks alter biochemical and
hematological parameters, with effects cot
strongly conclusive. However, histopathology
observations however suggested clear damage of
on the liver and lung of all the test groups with
youngest animals having more severe effects.

Although animals in different test groups show

some toxic effects; small animals of eight weeks
exhibit more effects with more severe lesions.
ACKNOWLEDGMENT
The research work was conducted during the

124

J. Toxicol. Environ. Health Sci.

Plate 1. Histopathology lesions. Photomicrograph of transversal section of New Zealand control rabbit liver from
Group A with distinct hepatocytes: hepatocellular central vein and the portal triads. H & E stain 10.

Plate 2. Histopathology lesions. Photomicrograph of transversal section of New Zealand test group with perilobular
toxic necrosis of the portal area. H & E stain 10.

Pouokam et al.

Plate 3. Histopathology lesions .Photomicrograph of the lung for control (H&E stain 40).

Plate 4. Histopathology lesions Group A (H&E stain 10) new Zealand rabbits; Observe the alveolar, a thickend
interalveolar septae with oedema fluid and widespread hemorrhage for test animals.

125

126

J. Toxicol. Environ. Health Sci.

internship programme in Toxicology, at the National

Veterinary Research Institute (NVRI), Vom Nigeria and
sponsored by the Africa Education Initiative (NEF), Mystic
Connecticut, USA. The authors are also grateful to
NVRIs research and laboratory staff for their support and
assistance.
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