Clinical and pathological features of Nerium oleander extract toxicosis in wistar rats
© Akhtar et al.; licensee BioMed Central. 2014
Received: 28 April 2014
Accepted: 12 December 2014
Published: 23 December 2014
Nerium oleander has been widely studied for medicinal purposes for variety of maladies. N. oleander has also been reported having noxious effects because of its number of components that may show signs of toxicity by inhibiting plasma lemma Na+, K+-ATPase. The present study was performed to scrutinize the toxic effect of N. oleander leaves extract and its clinical and pathological features in wistar rats.
Hematological analysis showed significant variations in RBCs count (P = 0.01), Hb (P = 0.001), Hct (P = 0.0003), MCV (P = 0.013), lymphocyte count (P = 0.015), neutrophil count (P = 0.003), monocyte count (P = 0.012) and eosinophil count (P = 0.006). Histopathological studies have shown that in T1 group noticeable infiltration of inflammatory cells was found with low level of vascular damage. In T2 group, increased proportion of binucleated and inflammatory cells, hepatic necrosis, widening of sinusoidal spaces and mild level of vascular damage was observed.
Taken together these findings we can conclude that N. oleander leaves extract significantly affects on experimental animals due to its toxicity. Efforts must be exerted to purify different chemical components from extract with no inflammation as this plant is utilized in folk medicine with narrow therapeutic indices.
KeywordsHistopathology Inflammation Toxicity Polycythemia Macrocytosis Neutrophilia
Nerium oleander commonly known as “Kaner”, belongs to the family Apocynaceae. It is native to Indo-Pak subcontinent, widely distributed in Mediterranean region, subtropical Asia, southern United States and many other warms areas  where it grows outdoors in parks, gardens and along roadsides by people who may not consider their toxic potential .
All parts of the plant are reputed as therapeutic agents and have been used in folklore in a variety of ailments including skin complaints, ringworm infections, opthalmia, cancer, epilepsy, eczema, malaria and gastrointestinal disturbances. Leaves and bark are also used as heart tonic, antibacterial, diuretic and anti-emetics [3–7].
On the contrary N. oleander has been regarded as poisonous plant due to a number of its components that may show signs of toxicity. Toxic exposure of humans and different species of domestic animals to N. oleander cardenolides occurs commonly throughout the geographic regions where this plant grows . The human mortality associated with ingestion of oleander is generally very low, but animals exposed to the plant are often found suddenly dead owing to cardiac dysfunction. N. oleander contains a mixture of very toxic cardiac glycosides of cardenolides, the most prominent of which are oleandrin and neriine [9, 10]. Cardiac glycosides of N. oleander cause poisoning by inhibiting plasmalemmal Na+, K+-ATPase . The plant has also been used for suicidal or murderous intention . Accidental and/or experimental N. oleander toxicities have been reported in cattle , horses , sheep , goats , donkeys , camels , cats , dogs , monkeys , budgerigars , geese , ducks , turkeys , toed sloths  and bears .
The intention of this study was to determine the toxic nature of N. oleander leaves extract and its clinical and pathological features in Wistar rats.
Healthy Wistar rats (about 175 ± 25 g) were arranged from the department of Zoology, University of the Punjab Lahore, housed in wire-bottomed cages in an animal room under standard conditions with 12-h light/dark cycles and at an ambient temperature of 22 ± 1°C, with fresh water and food pellets available ad libitum in compliance with the institutional guidelines and the study was approved by the ethics committee of the department of Zoology, University of the Punjab Lahore.
Dose preparation & administration
N. oleander leaves extract was prepared by boiling air-dried leaves in 0.9% NaCl solution (1:1, w/v) for 3 h by steam distillation. The extract was then filtered and used to the experimental animals. Rats were alienated into three groups, designated as Con group for control animals and T1 & T2 groups for experimental animals. T1 & T2 groups were given with ad libitum access to N. oleander leaves extract for 3 and 7 days respectively, whereas Con group was given with normal drinking water.
Blood & liver tissue sampling and processing
All the animals were anesthetized with intra-peritoneal injection of ketamine – distilled water mixture (1:1), (50 mg/ml of ketamine) and scarified. The dissections were done in aseptic maintained conditions to draw the blood through direct cardiac puncture and excise the liver out. The blood samples were collected in sterilized disposable syringes (Becton Dickinson, Private Ltd.), 2 ml of the blood was transferred to K3-EDTA coated vacutainers (Becton Dickinson, Private Ltd.) for complete blood count and liver of each animal, obtained after dissection was placed in Petri dish containing 0.9% saline, cut into 1 × 1 cm pieces and stored with 10% formalin in labeled glass bottles until.
Evaluation of hematological parameters
Complete blood counts were performed on the samples of control and treated animals, using an automated blood cell analyzer Sysmex XT-1800i (Japan). It utilizes the technology of fluorescent flow cytometry and hydrodynamic focusing. Fluorescent technology consistently differentiates normal white blood cells (WBCs), RBCs and platelets (plts) from abnormal populations, thereby decreasing the number of manual interventions.
Evaluation of histopathological variations
Small pieces of liver fixed in 10% formalin were dehydrated in ethanol (40%-100%), cleared in xylene and embedded in paraffin wax. Sections (5 μm thick) were cut and stained with haematoxylin and eosin and examined for histopathological changes under the microscope (Olympus VANOX). The microphotographs were taken using digital camera (Nikon SLR-D3000) camera at original magnification of 100X & 1000X.
The data were analyzed using Prism Graph pad 5 software (San Diego, CA). Statistical significance was calculated by means of one-way ANOVA test pursued by Tukey’s post hoc analysis to assess differences against the control group and p-values less than or equal to 0.05 were thought-out significant.
Consequence of N. oleander leaves extract on Red blood cells count & Red blood cell indices
Effect of N. oleander leaves extract on Red blood cells count & Red blood cell indices (Mean ± SEM) Con, control; T 1 , N. oleander oral intake for 3 days; T 2 , N. oleander oral intake for 7 days
7.810 ± 0.055
41.430 ± 0.185
14.500 ± 0.152
52.000 ± 1.000
8.600 ± 0.125*
45.330 ± 0.666**
16.070 ± 0.145**
62.500 ± 1.500*
7.960 ± 0.181
48.000 ± 0.577***
14.730 ± 0.218
54.670 ± 1.764
Consequence of N. oleander leaves extract on differential leukocyte count
Effect of N. oleander leaves extract on differential leukocyte count (mean ± SEM) Con, control; T 1 , N. oleander oral dose for 3 days; T 2 , N. oleander oral dose for 7 days
3.635 ± 0.115
0.580 ± 0.040
0.180 ± 0.020
0.016 ± 0.006
5.860 ± 0.330*
0.710 ± 0.010
0.265 ± 0.025
0.065 ± 0.005**
4.315 ± 0.215
2.650 ± 0.250**
0.055 ± 0.015*
0.010 ± 0.000
N. oleander is considered as poisonous plant. It contains components such as oleandrin and neriine that cause damage by inhibiting pasmalemmal Na+, K+-ATPase . In the present study the hematological and histopathological alterations were observed after N. oleander leaves extract administration in wistar rats. Hematological analysis indicates elevated level of RBCs, Hb, Hct and MCV in experimental animals. Increased number of red blood cells is an indication of polycythemia. In polycythemia, the levels of Hb, Hct, & RBCs are elevated when measured in the complete blood count (CBC), as compared to normal . Erythropoiesis occurs in the bone marrow. Erythropoietin (Epo) is one of the important hormones regulating this process . The majority of Epo is produced and released by kidneys (90%) and liver (10%) [27, 28]. Effect of extract on liver and kidney disturb the Epo expression directly or by disturbing other factors which are responsible for Epo expression. Under toxic conditions kidney release too much of protein (Epo) that enhance RBCs production. RBCs increase enhances the chance of clot formation, which can cause a heart attack or stroke. RBCs elevation is directly related to increase in Hb level . Hct, Packed cell volume is the volumetric content of the RBCs in the blood . Hct contents in the blood have been increased quit significantly (10% & 16%) in experimental groups; Hct can be increased in various physiological conditions such as in Dengue Shock Syndrome, polycythemia, myeloproliferative disorders, COPD chronic obstructive pulmonary diseases and hypoxia [31, 32]. The increase is in erythropoietin produced from the kidneys (specifically) is also the cause in increasing the Hct contents of the blood . The erythropoietin is also been designated as a positive acute phase protein due to its extremely high up regulation in initial stages of Acute phase response . The possible cause of increased Hct in the present study is increase in the MCV. The increase in MCV itself results in relative decrease in plasma . 20% increase in the MCV in T1 group blood analysis indicates a pathological state called as macrocytosis. The potent causes of the state are liver disease, compensated hemolysis, or folate deficiency .
Neutrophils and its derived cytokines play an essential part in the development and manifestation of inflammation. The stimulation of neutrophils can lead to the generation of oxygen derived free radicals also called reactive oxygen species (ROS) that cause further cellular damage. The formation of free radicals and cytotoxic oxygen metabolites probably impart a key role in various types of tissue degeneration and pathology such as aging, cancer and retinal degeneration [37, 38]. In the present study, significant elevation in the neutrophil count (neutrophilia) occurs with 357% rise in its level in T2 group as compared to control was may be due to the free radicals resulting from N. oleander leaves extract administration which caused liver injury and a proportion of these free radicals librated into the blood may also affect the circulating cells and induced a significant change in their number . Elevated number of lymphocytes secretes different derivatives. IL-5, a derivative of lymphocytes is responsible for the stimulation and activation of eosinophils . 47% increase in monocytes count indicates a state of stress response, necrosis, red cell regeneration and sarcoidosis [41, 42].
Tissue inflammation performs a crucial job in liver pathology. Experimental groups show infiltration of immune cells, widening of sinusoidal spaces, elevated level of binucleated cells and individual cell necrosis. It has been well established that free radicals elicit an inflammatory response at the site of injury . Toxic elements may induce pro-inflammatory cytokines by activating Kupffer cells . TNF-α, an important pro-inflammatory cytokine that is noticeably concerned in the progression or initiation of inflammatory response  and also stimulate the triggering of inflammatory mediators like IL-1, IL-6, TGF-β and prostaglandins that facilitate cellular infiltration, vacuolation and necrosis in the liver [46, 47].
Taken together these findings we can conclude that N. oleander leaves extract significantly affects on hematological and histopathological parameters due to its toxicity. Efforts must be exerted to purify different chemical components from extract with no inflammation as this plant is utilized in folk medicine with narrow therapeutic indices. Seeing that, its use is risky and should be cautiously researched.
The authors are thankful to the Vice Chancellor, University of the Punjab Lahore, Pakistan for providing financial support for the publication of this manuscript.
- Rashan LJ, Franke K, Khine MM, Kelter G, Fiebig HH, Neumann J, Wessjohann LA: Characterization of the anticancer properties of monoglycosidic cardenolides isolated from Nerium oleander and Streptocaulon tomentosum. J Ethnopharmacol. 2011, 134: 781-788. 10.1016/j.jep.2011.01.038.PubMedView ArticleGoogle Scholar
- O'Leary SB: Poinoning in man from eating poisonous plants. Arch Environ Hith. 1964, 9: 216-242. 10.1080/00039896.1964.10663823.View ArticleGoogle Scholar
- Bai L, Zhao M, Toki A, Sakai J, Yang XY, Bai Y, Ando M, Hirose K, Ando M: Three new cardenolides from methanol extract of stems and twigs of Nerium oleander. Chem Pharm Bull (Tokyo). 2010, 58: 1088-1092. 10.1248/cpb.58.1088.View ArticleGoogle Scholar
- Haq MM, Jabbar A, Rashid MA, Hasan M: A novel antibacterial and cardiac steroid from the roots of Nerium oleander. Fitoterapia. 1999, 70: 5-9. 10.1016/S0367-326X(98)00013-6.View ArticleGoogle Scholar
- Singhal KG, Gupta GD: Some Central Nervous System Activities of Nerium Oleander Linn (Kaner) Flower Extract. Trop J Pharm Res. 2011, 10: 455-461.View ArticleGoogle Scholar
- Suganya RS, Priya K, Roxy BM: Phytochemical screening and antibacterial activity from Nerium oleander and evaluate their plant mediated nanoparticle synthesis. Int Res J Pharm. 2012, 3: 285-288.Google Scholar
- Sugimoto H, Kataoka T, Igarashi M, Hamada M, Takeuchi T, Nagai K: E-73, an acetoxyl analogue of cycloheximide, blocks the tumor necrosis factor-induced NF-kappaB signaling pathway. Biochem Biophys Res Commun. 2000, 277: 330-333. 10.1006/bbrc.2000.3680.PubMedView ArticleGoogle Scholar
- Aslani MR: Poisonous plants of Iran and their effects on animals. 1st edition. 2004, Mashhad: Mashad university press, 210-212.Google Scholar
- Aslani MR, Movassaghi AR, Janati-Pirouz H, Karazma M: Experimental oleander (Nerium oleander) poisoning in goats: a clinical and pathological study. Iran J Vet Res. 2007, 8: 58-63.Google Scholar
- Langford SD, Boor PJ: Oleander toxicity: an examination of human and animal toxic exposures. Toxicology. 1996, 109: 1-13. 10.1016/0300-483X(95)03296-R.PubMedView ArticleGoogle Scholar
- Barbosa RR, Fontenele-Neto JD, Soto-Blanco B: Toxicity in goats caused by oleander (Nerium oleander). Res Vet Sci. 2008, 85: 279-281. 10.1016/j.rvsc.2007.10.004.PubMedView ArticleGoogle Scholar
- Lim DC, Hegewald K, Dandamudi N, Pettis L: A suicide attempt with an oleander cocktail. Chest. 1999, 116: 405-406.Google Scholar
- Aslani MR, Rezakhani A: A case report of oleander (Nerium oleander) intoxication in cattle. Int J Trop Agric. 2000, 18: 185-187.Google Scholar
- Hughes KJ, Dart AJ, Hodgson DR: Suspected Nerium oleander (Oleander) poisoning in a horse. Aust Vet J. 2002, 80: 412-415. 10.1111/j.1751-0813.2002.tb11000.x.PubMedView ArticleGoogle Scholar
- Smith PA, Aldridge BM, Kittleson MD: Oleander toxicosis in a donkey. J Vet Intern Med. 2003, 17: 111-114. 10.1111/j.1939-1676.2003.tb01332.x.PubMedView ArticleGoogle Scholar
- Vashishta MS, Singh RP: Camel diseases in India. 1977, Calcutta: Scientific Book Agency, 75-76. 1Google Scholar
- Burton LE, Picchioni AL, Chin L: Dipotassium edetate as an antidote in poisoning from oleander and its chief glycoside, oleandrin. Arch Int Pharmacodyn Ther. 1965, 158: 202-211.PubMedGoogle Scholar
- Humphreys DJ: Veterinary Toxicology. 1988, London: Bailliere Tindall, 1Google Scholar
- Schwartz WL, Bay WW, Dollahite JW, Storts RW, Russell LH: Toxicity of Nerium oleander in the monkey (Cebus apella). Vet Pathol. 1974, 11: 259-277. 10.1177/030098587401100307.PubMedView ArticleGoogle Scholar
- Shropshire CM, Stauber E, Arai M: Evaluation of selected plants for acute toxicosis in budgerigars. J Am Vet Med Assoc. 1992, 200: 936-939.PubMedGoogle Scholar
- Alfonso HA, Sanchez LM, Merino N, Gomez BC: Intoxication due to Nerium oleander in geese. Vet Hum Toxicol. 1994, 36: 47-PubMedGoogle Scholar
- Bardosi Z: Intoxication caused by oleander leaves. Magy Allatorv Lap. 1963, 18: 361-Google Scholar
- Shlosberg A, Ohad DG, Bellaiche M, Perl S: Monitoring of physiological and pathological changes in turkey poults fed leaves of potentially cardiomyotoxic Nerium oleander and Persea ameicana., 1st edn. 2004, Massachusetts: CABI PublishersGoogle Scholar
- Miller R: Oleander poisoning in a two toed sloth. J Zoo Anim Med. 1973, 4: 14-Google Scholar
- Jepson JH: Polycythemia: diagnosis, pathophysiology and therapy. II. Can Med Assoc J. 1969, 100: 327-334.PubMedPubMed CentralGoogle Scholar
- Aapro M, Jelkmann W, Constantinescu SN, Leyland-Jones B: Effects of erythropoietin receptors and erythropoiesis-stimulating agents on disease progression in cancer. Br J Cancer. 2012, 106: 1249-1258. 10.1038/bjc.2012.42.PubMedPubMed CentralView ArticleGoogle Scholar
- Eckardt KU, Ratcliffe PJ, Tan CC, Bauer C, Kurtz A: Age-dependent expression of the erythropoietin gene in rat liver and kidneys. J Clin Invest. 1992, 89: 753-760. 10.1172/JCI115652.PubMedPubMed CentralView ArticleGoogle Scholar
- Koury MJ, Bondurant MC: The mechanism of erythropoietin action. Am J Kidney Dis. 1991, 18: 20-23. 10.1016/S0272-6386(12)80285-X.PubMedView ArticleGoogle Scholar
- TOGNELLA S, BIGNOTTI G: Cardiovascular syndromes in polycythemia vera. Recenti Prog Med. 1964, 36: 43-82.PubMedGoogle Scholar
- Theml H, Heinz D, Haferlach T: Color Atlas of Hematology; Practical Microscopic and Clinical Diagnosis, 2nd revised edn. 2004, New York: Thieme New YorkGoogle Scholar
- Gross NJ: Chronic obstructive pulmonary disease. Current concepts and therapeutic approaches. Chest. 1990, 97: 19S-23S.PubMedGoogle Scholar
- Kociba GJ: Erythrocytes. Vet Clin North Am Small Anim Pract. 1989, 19: 627-635.PubMedView ArticleGoogle Scholar
- Ramadori P, Ahmad G, Ramadori G: Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6. Lab Invest. 2010, 90: 1306-1324. 10.1038/labinvest.2010.85.PubMedView ArticleGoogle Scholar
- Ramadori P, Sheikh N, Ahmad G, Dudas J, Ramadori G: Hepatic changes of erythropoietin gene expression in a rat model of acute-phase response. Liver Int. 2010, 30: 55-64. 10.1111/j.1478-3231.2009.02131.x.PubMedView ArticleGoogle Scholar
- Greer JP, John F, Lukens JN: Wintrobe's Clinical Hematology, 11th edn. 2003, Philadelphia: Williams & Wilkins PublishersGoogle Scholar
- Munker R: Modern Hematology; Biologyand Clinical Management. 2007, Totowa, New Jersey: Human Press Inc., 2View ArticleGoogle Scholar
- Hussain SP, Harris CC: Inflammation and cancer: an ancient link with novel potentials. Int J Cancer. 2007, 121: 2373-2380. 10.1002/ijc.23173.PubMedView ArticleGoogle Scholar
- Reznick AZ, Shehadeh N, Shafir Y, Nagler RM: Free radicals related effects and antioxidants in saliva and serum of adolescents with Type 1 diabetes mellitus. Arch Oral Biol. 2006, 51: 640-648. 10.1016/j.archoralbio.2006.02.004.PubMedView ArticleGoogle Scholar
- Doi K, Kurabe S, Shimazu N, Inagaki M: Systemic histopathology of rats with CCl4-induced hepatic cirrhosis. Lab Anim. 1991, 25: 21-25. 10.1258/002367791780808121.PubMedView ArticleGoogle Scholar
- Hamid Q, Azzawi M, Ying S, Moqbel R, Wardlaw AJ, Corrigan CJ, Bradley B, Durham SR, Collins JV, Jeffery PK: Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J Clin Invest. 1991, 87: 1541-1546. 10.1172/JCI115166.PubMedPubMed CentralView ArticleGoogle Scholar
- Fingerle G, Pforte A, Passlick B, Blumenstein M, Strobel M, Ziegler-Heitbrock HW: The novel subset of CD14+/CD16+ blood monocytes is expanded in sepsis patients. Blood. 1993, 82: 3170-3176.PubMedGoogle Scholar
- Fingerle G, Angstwurm M, Andreesen R, Ziegler-Heitbrock HW: Selective depletion of CD14+ CD16+ monocytes by glucocorticoid therapy. Clin Exp Immunol. 1998, 112: 501-506. 10.1046/j.1365-2249.1998.00617.x.View ArticleGoogle Scholar
- Padhy BM, Srivastava A, Kumar VL: Calotropis procera latex affords protection against carbon tetrachloride induced hepatotoxicity in rats. J Ethnopharmacol. 2007, 113: 498-502. 10.1016/j.jep.2007.07.007.PubMedView ArticleGoogle Scholar
- Shito M, Balis UJ, Tompkins RG, Yarmush ML, Toner M: A fulminant hepatic failure model in the rat: involvement of interleukin-1beta and tumor necrosis factor-alpha. Dig Dis Sci. 2001, 46: 1700-1708. 10.1023/A:1010653504568.PubMedView ArticleGoogle Scholar
- Hishinuma I, Nagakawa J, Hirota K, Miyamoto K, Tsukidate K, Yamanaka T, Katayama K, Yamatsu I: Involvement of tumor necrosis factor-alpha in development of hepatic injury in galactosamine-sensitized mice. Hepatology. 1990, 12: 1187-1191. 10.1002/hep.1840120518.PubMedView ArticleGoogle Scholar
- Basu A, Sen T, Ray RN, Chaudhuri AK: Hepatoprotective effects of Calotropis procera root extract on experimental liver damage in animals. Fitoterapia. 1992, 63: 507-514.Google Scholar
- Wang H, Wei W, Wang NP, Gui SY, Wu L, Sun WY, Xu SY: Melatonin ameliorates carbon tetrachloride-induced hepatic fibrogenesis in rats via inhibition of oxidative stress. Life Sci. 2005, 77: 1902-1915. 10.1016/j.lfs.2005.04.013.PubMedView ArticleGoogle Scholar
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