|Year : 2017 | Volume
| Issue : 1 | Page : 41-45
Effects of rauvolfia vomitoria on the cerebellar histology, body and brain weights of albino wistar rats
Aquaisua Nyong Aquaisua1, Christopher Chiedozie Mbadugha1, Enobong Ikpeme Bassey1, Moses Bassey Ekong1, Theresa Bassey Ekanem2, Monday Isaiah Akpanabiatu3
1 Department of Anatomy, University of Uyo, Uyo, Nigeria
2 Department of Anatomy, University of Calabar, Calabar, Nigeria
3 Department of Biochemistry, University of Uyo, Uyo, Nigeria
|Date of Web Publication||9-Aug-2017|
Aquaisua Nyong Aquaisua
Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, University of Uyo, Uyo
Source of Support: None, Conflict of Interest: None
Background: Rauvolfia vomitoria is a medicinal plant used in the treatment of hypertension and mental disorders. The effects of the ethanolic root bark extract of R. vomitoria were determined in this study.
Materials and Methods: Eighteen male and female young albino Wistar rats weighing 100–160 g were randomly assigned to three Groups 1, 2, and 3 of 6 rats each. The control (Group 1) received 0.1 ml of 20% solution of Tween 80, while Groups 2 and 3 received 200 mg/kg and 300 mg/kg of the root bark extract of R. vomitoria, respectively, for 14 days. The rats were sacrificed on the 15th day; the cerebellum of each rat was harvested, processed, and then stained using the hematoxylin and eosin method and immunohistochemical staining technique for glial fibrillary acidic protein (GFAP).
Results: The results showed swelling and atrophy of Purkinje cells, proliferation of reactive astrocytes and increased expression of GFAP, a significant reduction in the body weight of experimental animals, but no difference in brain weight compared with the control.
Conclusion: The ethanolic root bark extract of R. vomitoria has a deleterious effect on the cerebellum and body weight of young albino Wistar rats but does not affect brain weight.
Keywords: Body weight, brain weight, cerebellum, Purkinje cells, Rauvolfia vomitoria
|How to cite this article:|
Aquaisua AN, Mbadugha CC, Bassey EI, Ekong MB, Ekanem TB, Akpanabiatu MI. Effects of rauvolfia vomitoria on the cerebellar histology, body and brain weights of albino wistar rats. J Exp Clin Anat 2017;16:41-5
|How to cite this URL:|
Aquaisua AN, Mbadugha CC, Bassey EI, Ekong MB, Ekanem TB, Akpanabiatu MI. Effects of rauvolfia vomitoria on the cerebellar histology, body and brain weights of albino wistar rats. J Exp Clin Anat [serial online] 2017 [cited 2020 Dec 3];16:41-5. Available from: https://www.jecajournal.org/text.asp?2017/16/1/41/212639
| Introduction|| |
It is widely believed that over 80% of people in developing countries use herbal medicines as their first line of choice in the treatment of diseases (Moody, 2007). Despite advances in and availability of orthodox medicine, there is still a high patronage of traditional/herbal medicine for the treatment of mental and other disorders in Africa/Nigeria (Gureje et al., 1995). In Africa, in general, and in Nigeria, in particular, about 80% of the population resorts to herbal remedies (WHO, 2003).
R. vomitoria has been used over the years for the treatment of hypertension and mental disorders and it is a common herb used traditionally for psychiatric management in Nigeria (Akpanabiatu et al., 2006; Eluwa et al., 2008). It has been reported that all parts of R. vomitoria have a lot of medicinal values such as in curing of ailments such as malaria, typhoid, and jaundice among others (Fapojuwomi and Asinwa, 2013). It can be ground into powder and taken with pap and can be taken in the form of decoctions (Fetrow and Avila, 1999). Reserpine, an alkaloid extracted from this herb, is the first modern drug used in the treatment of hypertension (Reynolds, 1993; Onunkwo et al., 1998).
Various adverse effects including reduction of body weight, mild distortions of the cerebellum, and reduction in locomotion and exploratory behaviors have been attributed to administration of R. vomitoria to animals (Eluwa et al., 2008). The extract affects the cerebellar cytoarchitecture and neurobehavior; thus, the use of the drug should be limited to management of a diseased condition (Eluwa et al., 2008). It has been reported that high doses of ethanolic leaf and root extracts of R. vomitoria may be cardiotoxic to developing rats but that the root bark extract has more teratogenic potentials than the leaf extract (Eluwa et al., 2010). These previous findings have necessitated further evaluation of the effect of the plant on the histomorphology of the cerebellum of adult albino Wistar rats using the hematoxylin and eosin (H and E) staining technique and glial fibrillary acidic protein (GFAP) antibody staining protocol for immunohistochemistry as well as its effect on the body and brain weight of rats.
| Materials and Methods|| |
Eighteen male and female young albino Wistar rats weighing 100–160 g were obtained from the animal house of the Faculty of Basic Medical Sciences, University of Uyo, Uyo, Nigeria. Before the commencement of the experiment and throughout its duration, the animals were kept in the animal house in well-ventilated cages and maintained under normal room temperature. The animals were randomly assigned to three groups of six animals each, namely, Group 1 (control), group 2 and group 3 (experimental groups). All the animals were allowed access to growers mash (vital feeds from Grand Cereals Limited, Jos, Plateau State) and water ad libitum. The cages were cleaned and sawdust changed daily. All procedures involving animals in this study conformed to the guiding principles in the care and use of animals (American Physiological Society, 2002) and approval was granted by the animal use Ethical Committee of the Faculty of Basic Medical Sciences, University of Uyo, Uyo, Nigeria.
Collection and authentication of plant material
Fresh roots of R. vomitoria were obtained from a farmland in Ekpene Obo, Esit Eket Local Government Area of Akwa Ibom State of Nigeria. The roots were identified and authenticated by a botanist in the Botanical Garden, University of Uyo, Uyo, Nigeria. The roots were cleaned and the bark was obtained by scraping the roots with a knife. The air-dried bark was powdered in an electric blender and macerated in ethanol for 72 h at room temperature. The macerated powder was filtered and the filtrate concentrated in a water bath at 45°C to dryness. The extract was stored in a refrigerator until required.
Chemical tests for the screening and identification of bioactive constituents in R. vomitoria were carried out using standard procedures as described by Sofowora (1982), Trease and Evans (1989), Harborne (1993), and Sofowora (1993).
Acute toxicity study
The LD50 of the extract was determined using albino mice according to Lorke's method which is a limit dose test (Lorke, 1983).
Administration of the root bark extract and tissue processing
Group 1 (control group) received 0.1 ml of 20% solution of Tween 80 (Sigma-Aldrich Inc., P1379, Missouri, USA), while Groups 2 and 3 received daily doses of 200 mg/kg and 300 mg/kg of the extract, respectively, for 14 days. Two grams of the extract were dissolved in 20% solution of Tween 80 (20 ml) before administration. On the 15th day, all rats were weighed, anesthetized with ketamine, and perfused intracardially with 0.9% phosphate buffer solution, followed by 10% neutral buffered formalin. After sacrifice, the brains were carefully removed, weighed, and postfixed in 10% neutral buffered formalin for about 24 h. They were processed for paraffin blocks and sections were cut at 5 μ thickness. The tissues were stained using the H and E Technique of Drury and Wallington (1980) and the immunohistochemical technique of Faddis and Vijayan (1988).
The results were expressed as mean ± standard error of mean, analyzed using one-way analysis of variance followed by Student–Newman–Keuls post hoc test, and the difference between the mean of groups was considered significant at (P< 0.05) confidence level (Primer of Biostatistics: The Program © McGraw-Hill version 3.01) while histogram and error bars were prepared with MS Office Excel 2007.
| Results|| |
The phytochemical screening of the extract of R. vomitoria showed the presence of saponins, alkaloids, cardiac glycosides, carbohydrates, flavonoids, and terpenes while anthraquinones and tannins were not detected in the extract.
The LD50 of ethanolic root bark extract of R. vomitoria was calculated as 141.42 mg/kg through the intraperitoneal route and 1732.05 mg/kg through the oral route.
Body weight of the rats
There was a significant reduction in body weight of rats in experimental groups (P< 0.05) compared with the control group [Figure 1].
|Figure 1: Effect of ethanolic root bark extract of Rauvolfia vomitoria on body weight of young albino Wistar rats|
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There was no significant difference in brain weights between the experimental groups (P > 0.05) and the control group [Figure 2].
|Figure 2: Effect of ethanolic root bark extract of Rauvolfia vomitoria on brain weight of young albino Wistar rats|
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Histological and immunohistochemical findings
In H and E-stained sections, micrographs showed normal cerebellar architecture comprising the molecular layer, granule cell layer, and Purkinje cell layer, which showed intact Purkinje cells [Figure 3]. In immunohistochemical-stained sections, micrographs showed few (normal) astrocytes displaying positive but weak staining for GFAP [Figure 4].
|Figure 3: Micrograph of Group 1 (control) showing normal cerebellar architecture comprising the molecular layer, granule cell layer, and the Purkinje cell layer with intact Purkinje cells (H and E, ×400). MCL: Molecular layer, PC: Purkinje cell, GC: Granule cell|
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|Figure 4: Micrograph of Group 1 (control) showing few astrocytes displaying positive but weak staining for glial fibrillary acidic protein in the granule cell layer, within the periphery of the Purkinje cell layer. The astrocytes are not prominent in the molecular layer (GFAP, ×400). MCL: Molecular layer, PCL: Purkinje cell layer, GCL: Granule cell layer, ASC: Astrocytes|
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Group 2 (200 mg/kg)
In H and E-stained sections, micrographs showed swollen and atrophic Purkinje cells [Figure 5]. In immunohistochemical-stained sections, micrographs showed intense positive staining for GFAP and proliferation of reactive astrocytes [Figure 6].
|Figure 5: Micrograph of Group 2 (200 mg/kg) showing swelling of Purkinje cells (CS) and atrophy of Purkinje cells (AT) (H and E, ×400). CS: Cellular Swelling, AT: Atrophy of Purkinje cells|
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|Figure 6: Micrograph of Group 2 (200 mg/kg) showing intense positive staining for GFAP and proliferating reactive astrocytes surrounding the Purkinje cell layer (GFAP, ×400). ASC: Astrocytes|
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Group 3 (300 mg/kg)
In H and E-stained sections, micrographs showed swollen and atrophic Purkinje cells [Figure 7]. In immunohistochemical-stained sections, micrographs showed intense positive staining for GFAP and proliferation of reactive astrocytes [Figure 8].
|Figure 7: Micrograph of Group 3 (300 mg/kg) showing swelling of Purkinje cells (CS) and atrophy of Purkinje cells (AT) (H and E, ×400). CS: Cellular Swelling, AT: Atrophy of Purkinje cells|
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|Figure 8: Micrograph of Group 3 (300 mg/kg) showing intense positive staining for glial fibrillary acidic protein and proliferating reactive astrocytes surrounding the Purkinje cell layer (GFAP, ×400). ASC: Astrocytes|
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| Discussion|| |
Some adverse effects in animals have been linked to administration of the root bark extract of R. vomitoria (Akpanabiatu et al., 2009; Eluwa et al., 2010). The effects of the ethanolic root bark extract of R. vomitoria on the histology of the cerebellum, brain and body weight of young albino Wistar rats were investigated in this study.
The phytochemical screening of the root bark extract of R. vomitoria showed the presence of saponins, alkaloids, carbohydrates, cardiac glycosides, flavonoids, and terpenes. Tannins and anthraquinones were not detected in the extract. Olatokunboh et al. (2009) had reported the presence of alkaloids, saponins, carbohydrates, reducing sugars, and tannins in the extract of R. vomitoria. Another study by Sonibare et al. (2011) had reported the absence of tannins, cyanogenic glycosides, and anthraquinones.
The results of this study showed a significant difference between the body weight of the control group and experimental groups. The weight loss observed in the experimental groups may be as a result of gastric intolerance as well as drowsy, hypnotic, and sedative tendencies caused by reserpine, the major alkaloid in R. vomitoria (Eluwa et al., 2008).
There was no significant difference between the brain weight of the control group and experimental groups. According to Wilson (2001), test materials that alter body weight generally do not alter brain weight. Brain weight is less prone to be affected by changes in body weight and can be closely correlated with food consumption (Gill et al., 2015).
The sections obtained from the experimental groups indicated swelling and atrophy of Purkinje cells. These findings are suggestive of neuronal injury (Snell, 2010; Garman, 2011). When neurons react to injury, they become swollen and rounded off, the nucleus swells and is displaced toward the periphery of the cell. Cellular swelling, a sign of early injury, is due to disruption of energy-producing mechanisms and is accompanied by disruption of the sodium-potassium pump in the cell membrane. The resultant intracellular change allows the influx of sodium and water. Cellular swelling may be reversible if it disappears when the toxicant is removed (Richards, 2014).
The sections from the rats in the control group (Group 1) that were stained for GFAP revealed normal astrocytes. However, gliosis was observed in all the experimental groups. Proliferation of astrocytes is referred to as gliosis or astrocytosis (Snell, 2010). Astrocytes react to various neurodegenerative insults rapidly, leading to vigorous astrogliosis (Reier, 1986; Eng et al., 1992). After severe activation, astrocytes secrete various neurotoxic substances and express an enhanced level of GFAP, which is considered a marker protein for astrogliosis (Eng and Ghirnikar, 1994).
| Conclusion|| |
The ethanolic root bark extractof R. vomitoria induces injury in the cerebellum of young albino Wistar rats as evidenced by swelling and atrophy of Purkinje cells and gliosis. It also causes a loss in body weight but does not have an effect on brain weight.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Akpanabiatu M.I., Uboh F.E., Ekanem T.B., Umoh I.B., Eyong E.U., Ukafia S.O. (2009). The effect of interaction of Rauwolfia vomitaria
root bark extract with Vitamin E on rats' liver enzymes. Turk J Biol 33:189-94.
Akpanabiatu M.I., Umoh I.B., Eyong E.U. (2006). Influence of Rauwolfia vomitaria
root bark on cardiac enzymes of normal Wistar albino rats. Recent Prog Med Plants 14:273-8.
American Physiological Society. (2002). Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283:281-3.
Drury R.A., Wallington E.A. (1980). Carleton's Histological Techniques. 5th
ed., Vol. 1. Oxford University Press, New York, Pronto, p. 653-61.
Eluwa M.A., Idumesaro N.B., Ekong M.B., Akpantah A.O., Ekanem T.B. (2008). Effect of aqueous extract of Rauwolfia vomitoria
root bark on the cytoarchitecture of the cerebellum and neurobehaviour of adult male Wistar rats. Internet J Altern Med 6:8.
Eluwa M.A., Udoaffah M.T., Vulley M., Ekanem T.B., Akpantah A.O., Asuquo O.A., et al
. (2010). Comparative study of teratogenic potentials of crude ethanolic root bark and leaf extract of Rauwolfia vomitoria
(apocynaceae) on the fetal heart. North Am J Med Sci 2:592-5.
Eng L.F., Ghirnikar R.S. (1994). GFAP and astrogliosis. Brain Pathol4:229-37.
Eng L.F., Yu A.C., Lee Y.L. (1992). Astrocytic response to injury. Prog Brain Res 94:353-65.
Faddis B.T., Vijayan V.K. (1988). Application of glial fibrillary acidic protein immunohistochemistry in quantification of astrocytes in the rat brain. Am J Anat 183:316-22.
Fapojuwomi O.A., Asinwa I.O. (2013). Assessment of medicinal values of Rauwolfia vomitaria
(Afzel) in Ibadan Municipality. Greener J Med Sci 3:37-41.
Fetrow C., Avila J. (1999). Professional's Handbook of Complementary and Alternative Medicines. Springhouse Corporation, Pennsylvania, p. 4-7.
Garman R.H. (2011). Histology of the CNS. Toxicol Pathol 39:22-5.
Gill S., Kavanagh M., Cherry W., Barker M., Weld M., Cooke G. (2015). A 28 day gavage toxicity study in fischer 344 rats with 3-methylfuran. Toxicol Pathol 43:p. 221-32.
Gureje O., Acha R.A., Odejide O.A. (1995). Pathways to psychiatric care in Ibadan, Nigeria. Trop Med Int Health 47:125-9.
Harborne J.B. (1993). Phytochemistry. Academic Press, London, p. 89-131.
Lorke D. (1983). A new approach to practical acute toxicity testing. Arch Toxicol54:275-87.
Moody J.O. (2007). Traditional Medicine. Paper Delivered at the Mandatory Continuing Professional Development (MCPD) Programme, Module V, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria; 21-22, November.
Olatokunboh A.O., Kayode Y.O., Adeola O.K. (2009). Anticonvulsant activity of Rauwolfia vomitoria
(Afzel). Afr J Pharm Pharmacol 3:319-22.
Onunkwo G.C., Akah P.A., Udeala O.K. (1998). Stability of Rauwolfia vomitoria
tablets. Acta Pharm 48:63-9.
Reier P.J. (1986). Gliosis following CNS injury: The anatomy of astrocytic scars and their influences on axonal elongation. Astrocytes 3:263-24.
Reynolds B.J. (1993). Reserpine: The alkaloid from the Rauwolfia
plant. JAMA 162:325-469.
Richards I.S. (2014). Principles and Practices of Toxicology in Public Health. Jones and Bartlett Learning, Burlington, MA, p. 53.
Snell R.S. (2010). Clinical Neuroanatomy. 7th
ed. Wolters Kluwer, Lippincott Williams and Wilkins, Philadelphia, p. 61-2.
Sofowora A. (1982). Medicinal Plants and Traditional Medicine in Africa. 1st
ed. John Wiley and Sons, New York, p. 150-3.
Sofowora A. (1993). Medicinal Plants and Traditional Medicine in Africa. 2nd
ed. Spectrum Books Limited, Ibadan, p. 134-56.
Sonibare M.A., Lawal T.O., Ayodeji O.O. (2011). Antimicrobial evaluation of plants commonly used in the management of psychosis opportunistic infections. Int J Pharm 7:492-7.
Trease G.E., Evans W.C. (1989). A Textbook of Pharmacognosy. 13th
ed. Bailliere Tindall Limited, London, p. 19-21.
WHO. (2003). 56th
World Health Assembly Resolution (WHA 56.31) on Traditional medicine. Traditional medicine strategy. WHO, 2003, p. 2002-5.
Wilson A.G. (2001). Short term, subchronic and chronic toxicology studies. In: Hayes W., editor. Principles and Methods of Toxicology. 4th
ed. Taylor and Francis, Philadelphia, p. 80-106.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]