Short-term Toxicity Studies of Eupatorium adenophorum in Swiss Albino Mice

Abstract

Four groups of Swiss albino mice (male) were administered with vehicle (refined vegetable oil), 1/20 th , 1/10 th and 1/5 th of LD 50 doses of methanolic extract of Eupatorium adenophorum Spreng ; respectively for a period of 21 days. Mice fed with methanolic extract of Eupatorium adenophorum at a dose level of 752.2 mg/kg (i.e. 1/5 th LD 50) elicited hepatotoxicity and the animals had yellow discoloration of liver, subcutaneous tissue and musculature indicating jaundice. The sera samples revealed marked increase in bilirubin levels and activities of alkaline phos-phatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST). Histopathology of the livers of the group IV animals had focal areas of necrosis and bile duct proliferation. Elevation in plasma bilirubin concomitant with alterations in enzyme profile and histopathological lesions are consistent with liver injury and cholestasis.

Full text

Damodar Singh et al., Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 165-171
©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 165
Short-term Toxicity Studies of Eupatorium adenophorum in Swiss Albino Mice
Y. Damodar Singh*1, S.K. Mukhopadhayay2, M. Ayub Ali3, T.C. Tolenkhomba4 and M.A. Ayub Shah5
1Department of Veterinary Pathology, 3Department of Physiology & Biochemistry; 4Department of Animal Breed-
ing & Genetics and 5Department of Pharmacology & Toxicology College of Veterinary Sciences & A.H., Central
Agricultural University, Selesih, Aizawl, Mizoram, India
2Department of Veterinary Pathology, F/O Veterinary & Animal Sciences, WBUAFS, Kolkata-37, India
ABSTRACT
Four groups of Swiss albino mice (male) were administered with vehicle (refined vegetable oil), 1/20 th, 1/10th and
1/5th of LD50 doses of methanolic extract of Eupatorium adenophorum Spreng ; respectively for a period of 21
days. Mice fed with methanolic extract of Eupatorium adenophorum at a dose level of 752.2 mg/kg (i.e. 1/5th LD50)
elicited hepatotoxicity and the animals had yellow discoloration of liver, subcutaneous tissue and musculature
indicating jaundice. The sera samples revealed marked increase in bilirubin levels and activities of alkaline pho s-
phatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST). Histopathology of the livers of the group
IV animals had focal areas of necrosis and bile duct proliferation. Elevation in plasma bilirubin concomitant with
alterations in enzyme profile and histopathological lesions are consistent with liver injury and cholestasis.
Keywords: Eupatorium adenophorum; Hepatotoxicity; bilirubin; mice
INTRODUCTION
Eupatorium adenophorum Spreng (syn. Ageratina ade-
nophora, common name: Crofton weed; Sticky snake-
root), a native of Central America has appeared as a
major weed in several areas in different parts of the
world and has infested the grazing areas in the lower
and mid hills in the Himalayan region of India (Sharma
et al., 1998). E. adenophorum is widely growing shrub
in most parts of NE region, in general and Mizoram, in
particular. E. adenophorum is an important weedy co-
lonizer in early succession communities developing
after slash and jhum (shifting cultivation) at high eleva-
tions of North Eastern Hill Region of India (Kamakrish-
nan and Mishra, 2006 and Shah, 2007). It is a perennial
herb, nearly 1 meter high and erects (Mandal et al.,
2005). Grazing animals get accidentally exposed to the
plant under scarcity conditions. A considerable varia-
tion between the animal species exists in terms of sus-
ceptibility to toxicity due to E.adenophorum.
E. adenophorum has been investigated for its analges-
ic, anti-inflammatory and antipyretic properties (Bijar-
gi, 2009). The methanolic extract of E. adenophorum
exerted analgesic (Bijargi et al., 2009; Bijargi and Shah,
2010), anti-inflammatory (Bijargi and Shah, 2010, Bijar-
gi et al., 2010) and antipyretic (Bijargi and Shah, 2010)
effects in rats.
There is a lot of variation in the susceptibility of differ-
ent animal species to the noxiousness of E. adenopho-
rum (O’Sullivan, 1979 and Sharma, 1998). The content
of natural products in the plants is known to vary with
geographical region, soil and other environmental fac-
tors (Smith et al., 1994; Kaul and Vats, 1998). Con-
sumption of E.adenophorum by horses results in pul-
monary toxicity (Jones, 1954; O’Sullivan, 1979). Regular
ingestion of Eupatorium adenophorum [Ageratina ade-
nophora (Spreng.)] or Crofton weed causes chronic
pulmonary disease in horses mainly in Australia, New
Zealand, and the Himalayas (Oelrichs et al., 1995).
Toxicity due to consumption of this plant by other graz-
ing animals is not clear. However, no toxic effects were
seen in goats when E.adenophorum collected from
Nepal comprising up to 67% of their intake, was admi-
nistered (Neopane et al., 1992). Experimental feeding
of E.adenophorum plant growing in north-eastern India
to cattle caused anorexia, suspension of rumination
and photosensitization (Verma et al., 1987). In studies
with laboratory animals, mice were shown to be suita-
ble test animals, but in this species lesions occur in the
liver rather than the lungs. Exposure of mice to feed
containing E.adenophorum freeze-dried leaf powder
caused hepatotoxicity. In mice, hepatotoxicity involved
multiple areas of focal necrosis of the parenchyma a s-
sociated with degeneration and loss of epithelial cells
lining of the bile duct (Sani et al., 1992). E. adenopho-
rum leaf samples collected from Kangra Valley (India)
and partially purified extracts from leaf samples mixed
in the diet caused hepatotoxicity and cholestasis in rats
(Katoch et al., 2000 and Kaushal et al., 2001). We re-
port here the LD50 of E. adenophorum (methanolic ex-
tract) and its toxic effects in laboratory mice.
www.ijrpp.pharmascope.org
ISSN: 2231-010X
Research Article
* Corresponding Author
Email: drmaasvptcau@yahoo.co.in
Contact: +91-9436151974
Received on: 13-07-2011
Revised on: 27-07-2011
Accepted on: 28-07-2011

Damodar Singh et al., Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 165-171
166 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
MATERIALS AND METHODS
Collection, identification and extraction of the plant
material
The entire plant of Eupatorium adenophorum Spreng
was collected in April 2010 from the campus of the
College of Veterinary & A.H., Central Agricultural Uni-
versity, Selesih, Aizawl, Mizoram (India) and submitted
as a herbarium specimen for authentication to the Re-
gional Office, Botanical Survey of India (BSI), Shillong.
The BSI, Shillong has authenticated the plant as such by
Letter Reference No.BSI/ERC/Tech/2010/052, dated
27.04.2010.
Figure 1: Leaves and flowers of Eupatorium
adenophorum
The fresh leaves of the plant Eupatorium adenophorum
was carefully collected from the College campus,
washed gently till the leaves looked clean, mopped by
blotting paper and weighed. Then the plant leaves
were air dried under shade, protecting them from di-
rect sunlight for a period of one week. On complete
drying, whole of the leaves were ground to powder
with the instrument Willey grinder and sifted through
sieve number 22. The dry powder of Eupatorium ade-
nophorum was subjected to cold maceration technique
using methanol as solvent as follows:
The cold methanolic extract of E. adenophorum was
prepared as per standard method (Manjunatha et al.,
2005; Harborne, 1998). One hundred (100) grams of
whole plant powder was soaked in 500 ml of methanol
(1: 5 w/v) in a conical flask for 3 days with intermittent
stirring and at the end of 3rd day the content was fil-
tered with muslin cloth followed by Whatman filter
paper No-1. For complete extraction of the active prin-
ciples, this process was repeated three times using
fresh solvent on each occasion or until the colour of
the methanol became light. The filtrate obtained was
pooled and further subjected to vacuum evaporation
at 300C in a Rotary Evaporator and lyophilized for suc-
cessive 24 hours. Lyophilization was stopped when the
extract appeared sufficiently dry. Further the material
was stored at -45oC in deep freezer in air tight contain-
ers until use. The yield of the extract was 29.53 g per-
cent.
Preparation of oral suspension
The methanolic extract was found insoluble in water;
therefore, for different dose levels, a stock suspension
was prepared in refined vegetable oil and was diluted
with the vehicle (refined vegetable oil) immediately
before use for oral administration.
Experimental animals
In the present study, 50 male Swiss albino mice (Mus
musculus) of 25-30 g were obtained from the Colony
Stock of Laboratory Animal House, College of Veteri-
nary Sciences & A.H., Central Agricultural University,
Selesih, Aizawl, Mizoram. They were given a standard
pelleted diet and water ad libitum throughout the ex-
perimental period. A twelve-hour day and night cycle
was maintained in the animal house. The ambient
temperature and relative humidity during the experi-
mental period were 22-24oC and 65-70%, respectively.
The experimental protocol met regulatory guidelines
on the proper care and use of animals in laboratory
research.
Acute toxicity study
Thirty (30) male mice were randomly selected and di-
vided into six groups of five animals each. The animals
were fasted overnight. Group-I animals were orally
administered the vehicle (refined vegetable oil), while
the animals of Groups II-VI were given single doses of
methanol extract of E. adenophorum in progressively
increased manner (1350, 2025, 3050, 4575 and 6900
mg/Kg respectively) for determination of the acute
lethal dose (LD50). However, food and water were pro-
vided throughout the experiment. Immediately after
dosing, the animals were observed continuously for
the first 72 hours for mortality and any signs of overt
toxicity. The surviving animals were also observed up
to 14 days for signs of toxicity. The number of mice
that died within the period of study was noted for each
group, and subsequently the LD50 value calculated (Mil-
ler and Tainter, 1944). All animals that died during the
observation period and euthanatized mice were sub-
jected to necropsy.
Sub-acute toxicity study
Twenty (20) male mice were randomly divided into
four groups of five animals each. Animals of Group-I
served as vehicle (refined vegetable oil) treated con-
trols, while animals of Groups II, III and IV were admi-
nistered orally with the E. adenophorum extract at dai-
ly doses of 188 mg/kg (1/20th LD50), 376 mg/kg (1/10th
LD50) and 752 mg/kg (1/5th LD50) respectively for 21
days. Food and water were freely available during the
experiment. The animals in treated groups were ob-
served daily for physical and behavioural changes as
signs of toxicity. On termination of the experiment, all
the animals were weighed and then euthanized using
ether anesthesia. The blood samples were collected by
retro-orbital plexus into test tubes with the aid of a
capillary tube and then centrifuged at 4000 rpm for 10

Damodar Singh et al., Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 165-171
©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 167
minutes to separate the serum for estimation of blood
biochemical parameters. Gross lesions present in the
liver, lungs, kidneys, heart and spleen were recorded
and tissues showing lesions were fixed in 10% neutral
buffered formalin immediately for further histopatho-
logical examination.
Biochemical profile
Biochemical parameters; viz. blood glucose, serum
cholesterol, triglycerides, total protein, albumin, total
bilirubin, direct (Conjugated) bilirubin, alkaline phos-
phatase (ALP), alanine transaminase (ALT), aspartate
transaminase (AST), blood urea nitrogen (BUN) and
creatinine were estimated spectrophotometrically
(Spectroscan 2600 Chemito) using standard commer-
cial kits (Crest Biosystems, Goa, India).
Histopathological examination
The formalin-fixed tissues (2-3 mm thick) were taken,
washed overnight in running tap water and then dehy-
drated in ascending grades of alcohol starting from
50%, 70%, 90% and absolute alcohol I, alcohol II, alco-
hol III and finally cleared in cederwood oil or xylene.
These dehydrated tissue pieces were then embedded
in molten paraffin. Sections were cut at 3-5 μm thick
and stained with Mayer’s hematoxylin and eosin for
histopathological examinations (Bancroft and Stevens,
1980).
Statistical analysis
All the values were expressed as mean±SEM. Statistical
analysis was done using SYSTAT 6.0.1 version. The sta-
tistical significance of differences between the two
means was assessed by unpaired Student’s ‘t’ test. A
difference at P<0.05 was considered statistically signifi-
cant.
RESULTS AND DISCUSSION
Acute toxicity
Mice administered with E. adenophorum extract at the
dose level of 1350 mg/kg body weight showed no mor-
tality, while those at dose levels of 2025 and 3050
mg/kg body weight showed partial loss of appetite,
dullness and depression with 20% and 40% mortality
respectively in 24 hours. The dose level of 4575 mg/kg
body weight produced tremor, hypo-activity and 60%
mortality. However, the dose level of 6900 mg/kg body
weight had severe clinical signs and all animals died
within 4-6 hours.
The doses of LD50 study thus obtained were then plot-
ted on semi-logarithmic paper against the probit and a
best fitted linear scale was drawn. The oral acute LD50
of E. adenophorum extract was then determined from
the straight line and was found to be 3761 mg/kg body
weight (3364 ≤ 3761 ≥ 4157 mg/Kg with 95% confi-
dence). This LD50 value was lower than 5000 mg/kg
reported by other workers in mice with alcoholic ex-
tract of E. adenophorum Sprengel (Gao Ping et
al.,2005). However, methanolic extract of E. adeno-
phorum at 2000 mg/kg, die not produce any signs of
overt toxicity in rats (Bijargi et al., 2009). The differ-
ence in the LD50 values might be due to using of differ-
ent vehicle and also due to variation of geographical
region, soil and other environmental factors. This sug-
gests that the E. adenophorum plant growing in the
region is apparently more toxic.
Sub-acute toxicity
Clinical signs
The Group-I (vehicle control) mice remained normal
throughout the experimental period, while the animals
in Group-II (1/20th LD50 i.e. 188 mg/kg) showed a partial
Table 1: Changes in biochemical parameters of mice intoxicated with E. adenophorum methanolic
extract (21 days exposure).
Parameters
Group-I
(Vehicle
control)
Group-II
(188 mg/kg)
Group-III
(376 mg/kg)
Group-IV
(752 mg/kg)
Significance
Glucose (mg/dl)
100.43±3.44b
95.65±4.73b
113.29±3.57a
112.99±3.30a
**
Cholesterol (mg/dl)
181.01±6.51
192.64±8.15
186.63±4.72
185.21±3.43
NS
Triglycerides (mg/dl)
107.68±3.17
107.21±4.00
101.73±3.62
116.68±6.70
NS
Total protein (g/dl)
6.28±0.43
6.53±0.53
6.82±0.39
6.62±0.35
NS
Albumin (g/dl)
4.33±0.36
4.71±0.31
4.44±0.21
4.12±0.20
NS
Total bilirubin (mg/dl)
0.958±0.031d
1.27±0.058c
1.57±0.082b
3.47±0.078a
**
Conjugated bilirubin (mg/dl)
0.778±0.016d
1.17±0.038c
1.44±0.06b
3.25±0.06a
**
AST (IU/L)
33.80±1.49c
40.32±1.75c
68.40±2.08b
146.63±6.60a
**
ALT (IU/L)
43.65±2.45d
56.40±1.86c
91.29±4.25b
265.13±6.58a
**
Alkaline phosphatase (IU/L)
28.50±1.74c
37.05±1.86b
42.20±1.36b
112.14±3.06a
**
LDH (IU/L)
114.36±4.61c
103.13±4.75c
175.30±6.57b
453.94±9.56a
**
Urea (mg/dl)
43.05±1.43
44.17±1.91
42.32±1.06
40.89±1.32
NS
Creatinine (mg/dl)
0.486±0.035
0.534±0.013
0.496±0.025
0.530±0.029
NS
Values are Mean±SE. ** Significance at p ≤ 0.01; * p ≤ 0.05 and NS = Not significant

Damodar Singh et al., Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 165-171
168 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
loss of appetite, dullness and slight depression. The
Group-III animals (1/10th LD50 i.e. 376 mg/kg) became
dull, depressed and had rough hair coat after 10 days
of treatment. However, the animals in Group-IV (1/5th
LD50 i.e. 752 mg/kg) became dull, depressed and ano-
rectic within 10 days and leading to body weight loss
(data not shown). The mice from this group had rough
hair coat and appeared jaundiced when observed after
7 days of exposure. The ear pinnae and paws became
yellowish. The plants belonging to the genus Eupato-
rium are reported to be hepatotoxic and pneumotoxic
in horse (O’Sullivan, 1979)
Changes in the biochemical parameters
The effect of methanolic extract of E. adenophorum
(Spreng) on the biochemical parameters is summarized
in Table 1. The increase in serum glucose levels of
Groups III (376 mg/kg) and IV (752 mg/kg) animals
were highly significant (P <0.01) as compared to those
in Group-I (vehicle control) and Group-II (188 mg/kg).
No significant changes were observed in the levels of
cholesterol, triglycerides, total protein and albumin in
all other experimental groups. The total bilirubin levels
as well the conjugated bilirubin were much higher (P
<0.01) in the Group IV as compared to those in the
Groups II, III and the control. These observations pro-
vide the biochemical basis for the observed yellowish
discolouration of liver and the epidermis. The increase
in the bilirubin levels was more marked in the conju-
gated form which is characteristic of obstructive jaun-
dice and cholestasis (Cornelius, 1980). There was
marked increase (P<0.01) in the activities of aspartate
aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP) and lactate dehydro-
genase (LDH) in the animals in Group III and IV as
compared to those in Group-I (control) and Group-II.
The increase in the activity of transaminases is known
to be the indicators of degenerative changes in organs
or tissues like liver and myocardium (Kaneko, 1980;
Cornelius, 1989 and Neiger and Osweiler, 1989). In-
creased levels of transaminases and ALP activities are
also known to occur in a wide range of diseases of liver
like cholestasis, biliary obstruction and hepatic necrosis
(Tennant, 1997). The elevation of serum enzymatic
activities in the present study is attributable to E. ade-
nophorum -induced hepatic damage and/or necrosis as
confirmed from histopathological observations.
There were no changes in the levels of blood urea and
creatinine in all the E. adenophorum (methanolic ex-
tract)- treated groups as compared to the control indi-
cating that there was no nephrotoxic effect of metha-
nolic extract of E. adenophorum in mice when exposed
for 21 days.
Similar biochemical changes have been observed in the
plasma of rats exposed to leaf powder, methanolic
extract and partially purified fraction of E. adenopho-
rum (Sharma et al., 1998; Katoch et al., 1999; Kaushal
et al., 2000 & 2001 and Bhardwaj et al., 2001).
Figure 2: A) Livers of mice from groups I, II and III showing no appreciable cross changes and that of group
IV showing marked enlargement with yellowish discoloration. B) Postmartum examination of a mouse
from group IV showing yellowish discoloration of liver, subcutaneous tissue and musculature indicating
severe jaundice.

Damodar Singh et al., Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 165-171
©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 169
Gross and histopathological changes
Postmortem examination of the animals in the groups
I, II and III revealed no appreciable gross changes of the
liver and other visceral organs, while the animals of the
group IV which received 1/5th LD50 (752.2 mg/kg) of E.
adenophorum extract had yellowish coloration of liver,
subcutaneous tissue and musculature (Fig. 2 and 3) and
the urinary bladder was full of urine.
Histopathological studies also provided supportive evi-
dence for the biochemical analysis depicted by the
following photomicrographs. Figs. 3a and 3b showed
the normal architecture and mild degenerative
changes of liver with narrow sinusoidal spaces in
Groups I (control) and II (188 mg/kg) animals respec-
tively. Liver sections of Group III (376 mg/kg) animals
revealed mild to moderate bile duct proliferation and
focal areas of necrosis (Fig. 3c). No histopathological
lesions were observed in other tissues collected.
In Group-IV (752 mg/kg), the liver sections showed
dilatation of the bile ducts and proliferative changes
with infiltration of mononuclear cells (Fig.3d). The he-
patocytes around the bile ducts showed necrotic
changes as well as some focal areas of necrosis (Fig.
3d). Similar changes have been observed toxicity in-
duced by the whole leaf powder, methanolic extract
and partially purified fraction of E. adenophorum in
rats (Sharma et al., 1998; Katoch et al., 1999; Kaushal
et al., 2000 & 2001 and Bhardwaj et al., 2001). Bile
canalicular plasma membrane has been proposed as
the primary site of biochemical lesions in hepatotoxici-
ty induced by lantana toxins (Pass et al., 1978; Sharma
and Dawra, 1984). Other workers also observed dege-
neration of intrahepatic bile ducts and hepatocellular
necrosis following of administration of E. adenophorum
leaf powder to mice (Sani et al., 1989).
The present study shows that the toxicity of E. adeno-
phorum (methanolic extract) is dose dependant and
the dose level of 1/5th LD50 (752 mg/kg body wt) is
highly hepatotoxic in mice. Therefore, E. adenophorum
should be avoided from feeding to livestock as it could
be potentially toxic to higher animals too. Further long
term toxicity studies and on other toxicological aspects
on E. adenophorum are advocated.
ACKNOWLEDGEMENTS
The authors are thankful to the Dean, College of Vete-
rinary Sciences & A.H., Central Agricultural University,
Selesih, Aizawl, Mizoram for providing the facilities of
the College to conduct the experiment.
Fig. 3d
Fig. 3a
Fig. 3b
Fig. 3c
Fig. (3a) Control liver showing normal architecture (H&E x 200); (3b) Group-II liver showing mild dege-
nerative changes and narrowing of sinusoidal spaces of hepatocytes (H&E x 200); (3c) Group-III liver
showing mild to moderate bile duct proliferation

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170 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
REFERENCES
Bancroft, J.D. and Stevens, A. Theory and practice of
histologic technique. Churchill Livingstone, New York.
1980, p.89.
Bergmeyer, H.U. Methods in Enzymatic Analysis, Vol. 2,
Academic Press, New York. 1974.
Bhardwaj, R., Singh. A., Sharma, O. P., Dawra, R. K.,
Kurade, N. P. and Mahato, S. B. Hepatotoxicity and
cholestasis in rats induced by the sesquiterpene, 9-
oxo-10,11-dehydroageraphorone, isolated from Eu-
patorium adenophorum. J. Biochem. Mol. Toxicol.;
2001,15: 279-86.
Bijargi, S.R. Evaluation of analgesic, anti-inflammatory
and antipyretic properties of Eupatorium adenopho-
rum (Sticky Snakeroot) in rats. M.V.Sc. Thesis. Central
Agricultural University, Imphal (Manipur), India.
2009.
Bijargi, S.R. and Shah, M.A. Ayub. Analgesic and anti-
inflammatory properties of aqueous extract of Eupa-
torium adenophorum. Proc. International Conference
on Ethnoveterinary Practices. Centre for Ethnovete-
rinary Herbal Research & Training, TANUVAS, Than-
javur (Tamil Nadu).2010, p-96.
Bijargi, S.R., and Shah, M.A. Ayub . Antipyretic property
of Eupatorium adenophorum in milk-induce pyrexia
model in rats. Proc. International Conference on Eth-
noveterinary Practices. Centre for Ethnoveterinary
Herbal Research & Training, TANUVAS, Thanjavur
(Tamil Nadu).2010, p-96.
Bijargi, S.R., Ramdas, W., Hazarika, S., Gracia, L and
Shah, M.A. Ayub. Anti-inflammatory property of Eu-
patorium adenophorum methanolic extract in acute
and chronic models of inflammation in rats. Proc. In-
ternational Conference on Ethnoveterinary Practices.
Centre for Ethnoveterinary Herbal Research & Train-
ing, TANUVAS, Thanjavur (Tamil Nadu).2010, p-97.
Bijargi, S.R., Shah, M.A. Ayub and Lalmuanthanga, C.
Evaluation of analgesic property of ethanolic extract
of Eupatorium adenophorum (Sticky Snakeroot) in
rats. Proc. 42nd Annual Conference of Indian Pharma-
cological Society and International Conference on In-
tegrative Medicine. National Institute of Ayurveda
for Drug Development, Kolkata. Indian Journal of
Pharmacology.2009, 41 (Supplement 1): S148.
Bijargi, S.R., Shah, M.A. Ayub and Lalmuanthanga, C.
Acute toxicity studies on Eupatorium adenophorum
(Sticky Snakeroot) extracts. Proc. IX Annual Confe-
rence of Indian Society of Veterinary Pharmacology &
Toxicology, Anand Agricultural University, Anand.
2009, p-Tox-23.
Cornelius, C.E. Serum Enzyme Activities and Other
Markers for Detecting Hepatic Necrosis. 1989.
Gao Ping, Liu Yan-ping, Pan Wei-gao, Jiang De-hong,
Gan Ming-zhe, Qin Xiao-na, Liu Shi-gui. Toxicological
Study on Alcoholic Extracts of Eupatorium adenopho-
rum Sprengel. Sichuan Journal of Zoology. Sichuan
Journal of Zoology. 2005-01.
Harborne J.B. Phytochemical Methods A guide to
modern techniques of plant analysis. 3rd Ed. Springer
international Publication. 1998, p: 5.
Jones, N.L.C. Numinbah horse sickness. Institute of
Inspectors of Stock. NSW Yearbook. 1954, pp. 80–84.
Kamakrishnan, P. S. and Mishra, B. K. Population dy-
namics of Eupatorium adenophorum Spreng. during
secondary succession after slash and burn agricul-
ture (jhum) in north eastern India. Weed Resear.
2006, 22: 77-84.
Kaneko, J.J. Serum proteins and the dysproteinemias.
In Kaneko, J.J. ed. Clinical Biochemistry of Domestic
Animals. 3rd Ed. Orlando, Fla, American Press. 1980.
Katoch, R., Sharma, O.P., Dawra, R.K., Kurade, N,P,
Hepatotoxicity of Eupatorium adenophorum to rats.
Toxicon. 2000, 38: 309-314.
Katoch, R., Sharma, O.P., Dawra, R.K., Kurade, N.P.
Hepatotoxicity of Eupatorium adenophorum to rats.
Toxicon. 1999, 38: 309-314.
Kaul, V.K. and Vats, S. Essential oil composition of
Bothiochloa pertusa and phyletic relationship in
aromatic grasses. Biochemical Systematic Ecology.
1998,26: 347–356.
Kaushal, V., Dawra, R.K., Sharma, O.P., Kurade, N.P.
Hepatotoxicity in rat induced by partially purified
toxins from Eupatorium adenophorum (Ageratina
adenophora). Toxicon. 2001, 39: 615-619.
Mandal, S.K., Boominathan, R., Parimaladevi, B., De-
wanjee, S. and Mandal, S.C. Analgesic activity of me-
thanol extract of Eupatorium adenophorum Spreng.
leaves. Indian J. Exp. Biol. 2005, 43: 662-663.
Manjunatha, B.K, Vidya, S.M, Rashmi, K.V, Mankani,
K.L, Shilpa, H.J. and Jagadeesh, S. Evaluation of
wound healing potency of Vernonia arborea Hk. In-
dian J. Pharmacol. 2005, 37: 223-226.
Miller, L.C. and Tainter, M.L. Estimation of LD50 and its
error by means of log-probit graph paper. Proc Soc
Exp Bio Med. 1944, 57: 261.
Neiger, R. D., and Osweiler, G. D. Effect of subacute
low level dietary sodium arsenite on dogs. Fundam.
Appl. Toxicol. 1989, 13: 439-451.
Neopane, S.P., Shrestha, N.P., Gatenby, R.M., Aryal, I.K.
Performance of goats given different levels of ban-
mara (Eupatorium adenophorum) at Pakhribas Agri-
cutural Centre. PAC-Technical Paper. 1992No. 151: 1-
7.
O’Sullivan, B.M. Investigations into Crofton weed (Eu-
patorium adenophorum) toxicity in horses. Australi-
an Veterinary Journal. 1985. 62: 30-32.

Damodar Singh et al., Int. J. Res. Phytochem. Pharmacol., 1(3), 2011, 165-171
©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 171
O’Sullivan, B.M.,. Crofton weed (Eupatorium adeno-
phorum) toxicity in horses. Australian Veterinary
Journal. 1979, 55: 19-21.
Oelrichs, P.B., Calanasan, C.A., MacLeod, J,K,, Sea-
wright , A.A., Ng, J.C. The isolation of a compound
from Eupatorium adenophorum (Spreng.) (Ageratina
adenophora (Spreng.) causing hepatotoxicity in mice.
Nat Toxins. 1995, 3: 350-354.
Pass MA, Heath TJ The effect of Lantana camara on
intestinal motility in sheep. J Comp Path. 1978, 88:
149–156.
Reitman, S., and Frankel, S.A. Colorimetric method for
the determination of glutamic oxaloacetic and glu-
tamic pyruvic transaminase. Am J Clin Patho. 1957,
28: 56-63.
Sani, Y., Harper, P.A.W., Cook, R.L., Seawright, A.A., Ng,
J.C. The toxicity of Eupatorium adenophorum for the
liver of mouse. In: James, L.F., Keeler, R.F., Bailey,
E.M. Jr., Cheeke, P.R., Hegarty, M. (Eds.), Poisonous
Plants. Proceedings of the Third International Sympo-
sium 23–29 July 1989, Ames Iowa, Iowa State Unive-
risty Press. 1992, pp. 626–629.
Shah, M.A.Ayub Medicinal plants of North East region:
An overview. Proc. 7th Ann. Conf. of Indian Soc. Vet.
Pharmacol. Toxicol. and Natl. Symp. Ehtnopharma-
col. Clin. Vet. Practice and Role of Nanotech. Drug
Delivery Systems, Kerala Agricultural University, Poo-
kot (November 28-30, 2007). 2007, pp: 15-23.
Sharma, O.P. and Dawra, R.K. Effect of lantana toxicity
on canalicular plasma membrane of guinea pig liver.
Chem. Biol. Interact. 1984, 47: 369-374.
Sharma, O.P., Dawra, R.K., Kurade, N.P., and Sharma,
P.D. A review of the toxicosis and biological proper-
ties of genus Eupatorium. Natural Toxins. 1998, 6: 1-
14.
Sharma, O.P., Makkar, H.P.S., Dawra, R.K., and Negi,
S.S. Changes in blood constituents of guinea pigs in
lantana toxicity. Toxicol Lett. 1982, 11: 73-76.
Sharma, S., Sharma, O.P. Effect of lantadene A-induced
toxicity on enzymesassociated with cholestasis in
blood plasma of guinea pigs. Med Sci Res. 1999, 27:
157-158.
Smith, B.L., Seawright, A.A., Ng, J., Hertle, J.A., and Bos-
tock, P.D. Ptaquiloside in bracken fern (Pteridium
spp.) from eastern Australia and from cultivated col-
lections of bracken from world-wide sources. In: Co-
legate, S.M., Dorling, P.R. (Eds.). Plant associated
Toxins, Agricultural, Phytochemical and Ecological
Aspects, CAB International, Wallingford, UK. 1994,
pp. 45–50.
Tennant BC Hepatic function. In: Kaneko JJ, Harvey
JW, Bruss ML, editors. Clinical Biochemistry of Do-
mestic Animals, 5th edition. California: Academic
Press. 1997, pp 785-800.
Verma, A., Yadava, B.P.S., and Sampath, K.T. Possible
use of Eupatorium adenophorum Spreng. in animal
feeding. Indian Journal of Animal Nutrition. 1987, 4:
189–192.