Plant Archives Volume 20 No. 2, 2020 pp.

7840-7846 e-ISSN:2581-6063 (online), ISSN:0972-5210

ACUTE TOXICITY OF LEAVES ETHANOLIC EXTRACT OF NERIUM

OLEANDER (ALDEFLA) IN LOCAL IRAQI RABBITS (LEPUS CUNICULUS)

Duaa F. Hassan* and Falah M. Kadhim

Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine,
University of Baghdad, Baghdad. Iraq

Abstract
This study was conducted to evaluate the acute toxicity of the one of worldwide botanical Nerium oleander. First of all, the
leaves of plant had collected from Nerium oleander tree which grew up in Alneel distinct, Babil province, Iraq during August
2019. Leaves powder have been extracted by cold method of extraction by soaking in 95% ethanol. Median lethal dose (LD50)
of plant extract was performed in seven local healthy male Iraqi rabbits (Lepus cuniculus), up and down method have been
used for deriving LD50 after oral administration. Animals were observed during 24 hr of extract administration for any signs of
toxicity in all subjected animals, while necropsy finding and histopathology for several internal organs like lung, stomach and
heart have been studied in mortal animals only. The results showed, the yield extract of plant is 9.02%, while the LD50 of leaves
ethanolic extract is 232.23 mg/Kg.BW orally after 24 hours. Various clinical signs of toxicity were observed, briefly, salivation,
dyspenea, piloeraction, hind limbs extension, depression, convulsion, gasping etc. Necropsy finding included emphysema,
pulmonary congestion and bronchopneumonia, mild endocarditis, fibril layer on pericardial infiltrate and inflammatory cells
filaments, in stomach specifically, in glandular region there were severe erosion, lysis of mucosa extend to submucosa, most
glands in submucosa show severe necrosis.Various histopathological changes were observed such as pulmonary congestion,
thickening of alveolar walls, dilated pulmonary blood vessels, severe degeneration of bronchial epithelial mucosa and
necrosis, also there were pleuritis and pulmonary emphysema.Heart showed, infiltration and extension of inflammatory cells
to myocardium and acute pericarditis, thickened inflammatory layer replaced the flattened epicardium .whereas in stomach
there were severe atrophic necrosis and sloughing of mucosal epithelium, the sub mucosal glands severely degenerated and
necrotic.In conclusion: The ethanolic cold extraction is one of the useful method for obtaining significant yield extract of N.
Oleander leaves. The N. Oleander leaves ethanolic extract is considered severely toxic in rabbits after oral administration.
Key words: Nerium, oleander, lethal dose, ethanolic extract, rabbit, histopathology.

Introduction nausea, diarrhea, abdominal pain, arrhythmias and

hyperkalemia. The significant toxic impact of Nerium
Nerium oleander known by local Iraqi name Aldefla,
oleander poisoning is cardiotoxicity (ventricular
is a toxic plant of the Apocynaceae family to humans,
arrhythmia, bradycardia, tachycardia (Farkhondeh et al.,
animals and insects (Khordadmehr and Nazifi, 2018). It
2020). Nerium Oleander poisoning is not uncommon in
is a small tree evergreen with a wide distribution in world.
domestic animals and man and there have been some
Its flowers grow in clusters in incurable branches and cases of dangerous toxicosis in children and adults.
show various colors including yellow, pink, red, peach Nerium oleander is a plant that is often grown in gardens
and white. All parts of the plant can be very toxic to and public areas. Various Pharmacological effects of
insects, several of animals and humans(Farkhondeh et Nerium oleander including antinociceptive, anti-
al., 2020; Ghurghure et al., 2019). Children are very inflammatory and anticancer activity were reported (Salih
susceptible to the N. oleander toxicity. Unintentional 2017). The extracts of many parts of Nerium oleander
ingestion of plant in children and use of this plant for Leaves are used as antidiabetic remedy in the traditional
suicide are two main lead to N. oleander toxic in the medicinal systems of many parts of the world (Dey et
world. The important clinical finding consists of vomiting, al., 2019). Nerium oleander is used to produce
*Author for correspondence : E-mail: dr.duaa.pharma@gmail.com cardiotonic medicine, this plant used as an anti-cancer
 Acute Toxicity of Leaves Ethanolic Extract of Nerium oleander (Aldefla) in Local Iraqi Rabbits 7841

medicine and alternative medicine for many causes Materials and Methods
(Azzalini et al., 2019), but the potential toxic effects of Leaves of plant Nerium oleander are collected from
all parts of the shrub either fresh or dried on animal and Babil province, A.L. Neel at 2019 August (Fig. 1). The
human body were documented (Serra, 2016). It contains plant is 3 meters in long with dark-green leathery
numerous toxic compounds. The major toxic components lanceolate leaves and has white flowers. It is dried at
found are the cardiac glycosides oleandrin and neriin room temperature with opened windows and door for
(Abdou, Basha and Khalil, 2019). one week, then grind by using electrical grinder and kept
Aldefla is toxic when consumed, the cause of toxicity in the tightly stopped glass bottle and kept in the
is mostly due to the presence of cardiac glycoside refrigerator under 4oC till used. The powder of Nerium
particularly oleandrin, firstly it has a digitalis action. Many oleander leaves is extracted by ethanol (Chem. Lab NV,
attempts are made to use it as cardiac tonic but not be Belgium). Fifty g of plant dried powder have been soaked
successful due to narrow therapeutic index. Other toxic into 500 ml of 95% ethanol with continuous stirring for
constituents of this plant is cardinolides and niriine which two hours by using magnetic stirrer machine and kept in
are glycosides, second the bark of the plant have rosagenin conical flask in ice bath for 24 hours in refrigerator, then
which has a strychnine action, three the signs of toxicity filtered by using piece of gauze, after that the filtrate
and the causes of death is the similar for other cardiac filtered one more by using apparatus of Buchner through
glycosides as digoxin (Li et al., 2020). 0.4 millipore filter under negative pressure (27). The yield
The toxic effects of plant or their active alkaloids extract was calculated by applying the following equation:
lead to infiltration of cells with hemorrhage and severe
negative variations in the lung, induce lesions and
infiltration of inflammatory cells into the portal places (Banso and Adeyemo, 2006 ).
with scattered necrosis of hepatocytes in the liver, heart
toxicity of the plant in the cardiac were included, induced • Animals: A total of seven male local Iraqi rabbits
multiple degrees of hemorrhage, myocardial degeneration (Lepus cuniculus) purchased from the animal house of
and necrosis. It also lead to arrhythmia, sinus bradycardia college of veterinary medicine, university of Baghdad,
and in ECG records prolonged P-R interval. The toxic their weight range is 1000-1700 gm and age 6-12 month,
effects of N. oleander are typically related to its the animals raised in the animal house of college of
inhibitory effects on the Na-K ATPase pump in the cellular veterinary medicine, university of Baghdad for four weeks
membrane (Karthik et al., 2020). to acclimatization under suitable condition of temperature
25±1oC and dark/light cycle 12/12 hours, the animals fed
Due to wildly distribution of this plant which is may ad labium with standard pellet, green grass and water in
contribute into various toxicosis in animals which may air-conditioned room.
grazing and in human regarding un-intentional ingestion.
We are planned to assessment its acute toxicity which is Assessment of Median Lethal Dose (LD50)
considered the important tool for assessment the risk of The median lethal dose of leaves ethanolic extract
any chemical, biological and botanicals. of N. oleander is assessed by up and down method
(Dixon, 1965) briefly, N. oleander leaves extract was
given orally by gastric gavag to the animals at range of

Fig. 1: Nerium Oleander (Aldefla) from AL Neel district, Babel,

Iraq. Fig. 2: Ehanolic extract of N. oleander leaves extract.
7842 Duaa F. Hassan and Falah M. Kadhim

Fig. 3: Lung of rabbit after administration of Nerium oleander

leaves by ethanolic extract at dose 240 mg/kg Bw. Fig. 5: Stomach of rabbit after administration of Nerium oleander
leaves by ethanolic extract at dose 240 mg/kg Bw.
doses between 150-240 mg/kg.BW and the difference in
doses was 30mg/kg.BW. LD50 was calculated after 24 2), it is yield about 54.12g from 600 g crud powder which
hours observation of dead and life animal by applying the is representing 9.02%.
following equation: The oral median lethal dose (LD50) of N. oleander
LD50= xf + kd ethanolic extract that found by up and down method is
232.23 in local male Iraqi rabbits (Lepus cuniculus) for
Xf = last dose administrated, 24 hr (Table 1).
K= constant, d= difference between dose LD50 = xf +kd
• Pathology: Each dead rabbit has been dissected LD50 = 210 +(0.741*30 )
as soon as for necropsy finding and microscopic lesion
of several organs like heart, lung and stomach. The animal LD50 = 232.23 mg /kg.BW orally in rabbit after 24
is examined grossly after 10 minute from death. The hours.
organs preserved in 10% formalin buffer for fixation Kd = 0.741
before embedding the tissues in paraffin block, 3-10 • Clinical findings: The morbid rabbits showed
micron thickness of histological sections were prepared various signs of poisoning like depression, pawing the
by microtome. All histological sections have been stained ground, anorexia, piloeraction, dyspeniea, slightly watery
by hematoxylin and eosin stain (H&E) (AL-Naqeeb and salivation, extended of hind limb while the fore limb is
Yousif, 2011). unaffected, tachypeniea, froth in the mouth. Table 2,
shows the most important symptoms with the timing of
Results
their emergence and disappearance within 24 hours after
Extraction yield of N. oleander leaves ethanolic the dose with the leaves ethanolic extract of N. oleander
extract is dark green in color, amorphous crystals (Fig.

Fig. 6: Section of lung shows: mild interstitial pneumonia (In),

Fig. 4: Heart of rabbit after administration of Nerium oleander pulmonary emphysema (E) & congestion (C). H&E
leaves by ethanolic extract at dose 240 mg/kg Bw. stain. 40x
 Acute Toxicity of Leaves Ethanolic Extract of Nerium oleander (Aldefla) in Local Iraqi Rabbits 7843

Table 1: The outcome of median lethal dose (LD50) of orally administration of stomach) and showed the submucosal
ethanolic extract of Nerium oleander leaves in Local Iraqi rabbit (Lepus glands severely degenerated and
cuniculus) calculated by up and down method. necrotic (Fig. 8).
Initial dose Different between Last LD50 orally mg/kg
mg/kg .BW doses mg /kg .Bw dose
Outcomes*
.BW after 24 hr
Discussion
150 30 210 OOOXOXO 232.23 Acute toxicity defines the adverse
* X= Dead animal; O= alive animal effects of a substance that outcome
for different doses. either from a single exposure or from many exposures in
• Necropsy Finding: The necropsy finding of dead a short period of time (typically less than 24 hours). To
animals showed, there were severe emphysema, be pronounced as acute toxicity, the adverse effects
pulmonary congestion and bronchopneumonia (Fig. 3), should happen within 14 days of the administration of the
while in heart we are observed was clots, fibrinous substance, also refer to poisonous state (Hatif, Kafi and
exudate, mild endocarditis, fibril layer on pericardial Alkhayyat, 2010). Nerium Oleander leaves decoction
infiltrate and inflammatory cells filaments, (Fig. 4) and, give to male rat injected intramuscularly in both hind limbs
in stomach specifically, in glandular region there were in two groups,group one injected 5 ml/kg and group two
severe erosion, lysis of mucosa extend to submucosa most injected 10 ml/kg. The results refer to both doses can
glands in submucosa show severe necrosis (homogenous induce acute phase condition (Abbasi et al., 2017;
and structure less) (Fig. 5). Klaassen and toxicology, 1986).
Histopathological finding Median lethal dose (LD50) is the dose essential to
The histopathological finding of organs rabbit after kill half the members of a examined population after a
administration of ethanolic extract of Nerium Oleander specified test duration. It is a tool to measure the acute
leaves at dose 240 mg/kg Bw. orally lungs in rabbit which toxicity of a chemical active ingredient. Also use to detect
have been observed are pulmonary congestion, thickening the toxicity of plant, to ass therapeutic dose of plant from
of alveolar walls, dilated pulmonary blood vessels filled through test LD50 (Saliem, 2010). The estimated LD50 of
with blood and few inflammatory cells, severe ethanolic extract of Nerium Oleander leaves in current
degeneration of bronchial epithelial mucosa and necrosis, study is 232.23 mg /kg.Bw orally in rabbit could be
also there were pleuritis and pulmonary emphysema (Fig. scheduled in the grade 4 severely toxic agent according
6). Heart showed, infiltration and extension of to classification dependent by (Hayes and Loomis, 1996).
inflammatory cells to myocardium and acute pericarditis, The clinical signs that have been observed on exposed
thickened inflammatory layer replaced the flattened rabbit included depression, anorexia, piloeraction,
epicardium (Fig. 7). Whereas the histopathological finding dyspeniea, slightly watery salivation, extended of hind
in stomach included severe atrophic necrosis of mucosal limb while the fore limb is unaffected and tachypeniea.
epithelium, complete sloughing in other section, the sub These clinical signs are in agreement with those observed
mucosal glands severely degenerated and necrotic of after one hour in mice exposed ethanolic extract of
Nerium oleander leaves at of 520 mg/kg body weight

Fig. 7: Section of heart shows: mild endocarditis with clot (c), Fig. 8: Section of stomach shows: (E) erosion and, (n) sever
fibrin exudate and inflammatory cells (red arrow). H&E degeneration and necrosis of gastric glands (Arrows).
stain. 40x. H&E stain. 40x.
7844 Duaa F. Hassan and Falah M. Kadhim

Table 2: Signs of poisoning and mortality outcome that developed according to different doses of N. Oleander leaves Ethanolic
Extract in Local Male Iraqi Rabbits (Lepus cuniculus) according to Up and Down method.
Dose Results
Clinical signs during 24 hrs Time of signs appeared
mg/kg BW (O, X)
depression, anorexia, piloeraction, dyspeniea,
150 mg slightly watery salivation, extended of hind limb After five hours from administration O
while the fore limb is unaffected, tachypeniea.
depression, anorexia, piloeraction, dyspeniea,
180 mg slightly watery salivation, extended of hind limb After three hours from administration O
while the fore limb is unaffected, tachypeniea.
depression, anorexia, piloeraction, dyspeniea,
210 mg slightly watery salivation, dragging of hind limb After three hours from administration O
while the fore limb is unaffected, tachypeniea.
sever convulsion, gasping,slightly watery
240 mg salivation, piloeraction,extruded of eye ball, head After 15 minute from administration X
down, incordination, circuling around itself .
depression, anorexia, piloeraction, dyspeniea,
210 mg slightly watery salivation, dragging of hind limb After two hours from administration O
while the fore limb is unaffected, tachypeniea.
depression, anorexia, piloeraction, dyspeniea,
210 mg slightly watery salivation, dragging of hind limb After two hours from administration O
while the fore limb is unaffected, tachypeniea.
was administered orally, the signs of toxicosis have leaf extract Nerium oleander in rabbit which was 157.37
appeared, abdominal pain, frequent urination, weakness, mg / kg B. wt, but fall into the same grade of toxicity (Al-
diarrhea, depression, convulsive movement and death Badrani, Rhaymah and Al-Farwachi, 2008). While the
(Saliem, 2010). While clinical signs of aqueous extract of median lethal dose of leaves Nerium oleander ethanolic
Nerium oleander in female goat that appeared after 1 extract is 520 mg/kg body weight were giving orally to
hr from receiving the extract were abdominal pain, male mice (Saliem, 2010). Whereas several studies found
convulsion, ruminalatony, depressin, convulsion and death the orally LD50 in mice of hexane leaf extracts of N
(Aslani et al., 2007). The clinical signs that observed on oleander has red, pink and white flowers was 325, 300
rabbits of our study were not fully agree with a study and 350mg/kg respectively (6). The oral LD50 of aqueous
conducted in dogs have been administered orally extract of Nerium oleander female goat is 110mg/kg
0.25g.kg-1 of fresh ground leaves of Nerium oleander, BW (12). The LD50 that has been found in rabbits in
when several dogs died after the ingestion, but all showed current study is disagree with the LD50 of N. oleander
signs of poisoning such as vomiting, diarrhea, nausea, leaves collected from Amiriya /Baghdad-IRAQ in summer
apathy, conjunctiva congestion, dehydration, abdominal and winter, which were extracted by hexane and derived
pain, tremors, diarrhea, loss of appetite and tenesmus. by “up and down” method were 94.36, 79.75 mg/kg BW
Loss of appetite was prominent in 30% of the dogs even respectively (Salih and Alkhayyat, 2016), on the other
after the treatment. The first obtainable clinical sign was hand the oral LD50 of dried Nerium Oleander leaves
vomiting, within 27- 75 minutes of administration of ground was 110 mg /kg.BW in sheep which possessed clinical
green leaves. Second presented sign was loss of appetite, signs of poisoning appeared too early after 30 minutes of
after the intoxication and after the treatment, with 40% administration (Aslani et al., 2004), whereas the oral
of the animals having these signs for as long as 12 hours LD50 of N. oleander leaves after a simple extraction
after giving (Camplesi et al., 2017). The clinical signs from biological samples, for example. in cattle is 50 mg/
appeared in sheep after 30 minute from take dried Nerium kg.BW. The leaf fragments found in the rumen contents
Oleander leaves were decrease heart rate (cardiac were small and not simply isolatable from the rest of the
pauses), tachyarrthemia, mild to moderate tympany, contents. Though, there were leaves in the forage that
abdominal pain, polyuria and pollakiuria (Aslani et al., were clearly distinct by the direct examination. Oleander
2004). poisoning due to active components contained in the plant
The estimated LD50 of ethanolic extract of Nerium are cardiac glycosides belonging to the cardenolides that
oleander leaves in current study is (232.23 mg /kg.Bw are very toxic to many species, from human to insects
orally in rabbit is more than the LD50 s/c injected aqueous (Rubini et al., 2019).
 Acute Toxicity of Leaves Ethanolic Extract of Nerium oleander (Aldefla) in Local Iraqi Rabbits 7845

The toxicity of Nerium oleander due to the present were collected in late May 2014 from University of Sistan,
of the highest concentrations of cardiac glycosides are in Baluchestanin southeast of Iran (Einali et al., 2018) and
the seeds and roots of the common pink Nerium oleander, the our result better than this studies by the non-polar
while the highest component of oleandrin are found in solvents via petroleum ether (2.87%) was less as
the leaves of the plant. Furthermore, N. oleander with compared to the yield obtained from the polar solvents
red to pink flowers has higher concentrations of cardiac by ethanol and water 5.7% and 6.8% respectively. The
glycosides than Nerium oleanders with white flowers fraction yield obtained from the successive extraction
(Khordadmehr and Nazifi, 2018). So the thought of using the solvents petroleum ether, ethanol and aqueous
decrease in LD50 rate resulted from administration N. extract obtained by maceration, from root of Nerium
oleander leave ethanolic extract to rabbits in current Oleander (Kawalekar et al., 2012), while disagree with
study comparable to that found in different literature may the yield of extract 26%, 18% by Soxhlet method by the
be due less abundant of oleanrinin leaves of plant with two hydro-methanoic and hydro-ethanolic extracts
white flower which has been used. Nerium oleander respectively of Nerium oleander leaves 20g of fresh
Leaves have high amount of cardiac glycoside and leaves extract by ether petroleum and then dry for 10
flavonoids compounds from methanolic extract of flower minutes at room temperature, after that solute is placed
(Vu, Bui and Nguyen, 2016). Oleander toxicity is a in Soxhlet (Malika, Badiaa and Farida, 2016). The other
common problem in several parts of the world. Oleander studies showed extraction yields for every kilogram of
poising due to oleandrin (the chief cardiac glycoside of leaves Nerium oleander by ethanolic extract was 1.03%
oleander) and neriin, which lead damage by inhibiting the (de Melo et al., 2020).
plasmalemmal Na+/K+ ATPase (Abdou, Basha and Khalil,
2019). Oleander contains cardiac glycosides called Conclusion
cardinolide. Cardiac glycoside may cause hyperkalemia The ethanolic cold extraction is one of the useful
because of ability to inhibit the Na+K+-ATPase pump, method for obtaining significant yield extract of N.
leading to increased intracellular sodium and calcium and Oleander leaves. The N. Oleander leaves ethanolic
decreased intracellular potassium (Salih, 2017). The extract is considered severely toxic in rabbits after oral
concentration of oleandrin in N. oleander tissues is about administration.
0.08% (Salama et al., 2019). Nerium oleander is
cardiotoxic plant and Its toxicity is due to the content of Conflict of interest
cardioactive glycosides, specially oleandrin, found The authors declare that they have no conflict of
throughout the plant (Botelho et al., 2018). The ethanol interest
extracts of Nerium oleander leaves which have the
highest amount of entirephenolics and flavonoids, Acknowledgments
appeared the highest antioxidant and cause toxicity of The authors are highly appreciable to department of
plant (Einali et al., 2018). Oleander poisoning due to active physiology, biochemistry and pharmacology, College of
components contained in the plant are cardiac glycosides Veterinary medicine University of Baghdad for providing
belonging to the cardenolides that are very toxic to many equipments.
species, from human to insects (Rubini et al., 2019).
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