Continental J. Pharmacology and Toxicology Research 4 (1): 30 - 34, 2011 ISSN: 2141 4238 Wilolud Journals, 2011 http://www.wiloludjournal.

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TOXICOLOGICAL EFFECTS OF EXTRACTS OF THE LEAVES OF Scoparia dulcis ON THE BRAIN OF RATS Esume C.O Department of Pharmacology, Delta State University Abraka and Department of Medicine, Central Hospital, Warri ABSTRACT BACKGROUND: Scoparia dulcis is a widely used medicinal plant with scanty information on the toxicity profile. This work was conceived to ascertain the toxic effect on the central nervous system following acute exposure. METHODOLOGY: Varying doses of the methanol and water extracts that are higher than the LD50 were administered intraperitoneally into 4 groups of 5 rats each and their brain dissected out and histopathological analysis carried out on them. RESULTS: The results showed that at toxic doses, the extracts have potent pro inflammatory effects with tendency to cause cerebral edema. DISCUSSION: This is due to the derangement in cellular metabolism resulting in inadequate functioning of the sodium and potassium pump in the glial cell membrane. As a result there is cellular retention of sodium and water. There are swollen astrocytes in grey and white matter. Cytoxotic edema is seen with various intoxications. In clinical cases of toxicity, where it is confirmed that the offending plant is Scoparia dulcis, treatment should be directed at management of cerebral edema, as this is one of the most affected organ in the body. KEYWORDS: cellular metabolism, histopathological analysis, rats, Scoparia dulcis, treatment, INTRODUCTION Scoparia dulcis is an erect annual herb with serrated leaves, producing white flowers and measuring up to a half meter in height when fully grown (Orhue et al, 2004). This plant is found in abundance in South America and the Amazon rainforest and is widely distributed in many tropical countries in the world, Nigeria inclusive. The aerial parts, the leaf and the root of this plant have been traditionally used in herbal medicine for their analgesic, antibacterial, antifungal, antiherpetic, antinflammatory, antiseptic, antispasmodic, antiviral, cytotoxic, emmenagogue, emollient, febrifuge, and hypotensive effects (Freire et al, 1993). It is sometimes added to foods meant for pregnant and nursing mothers for medicinal purposes (Okwu 1999, 2001). Despite the wide usage of Scoparia dulcis as a medicinal plant especially in the treatment of hypertension and diabetes, information on the acute, subacute and chronic toxicity is very scanty. This work intends to study the acute toxicological effects of aqueous and methanol extracts of Scoparia dulcis in the brain of rats using Histopathological methods. METHODS The leaves of the plants were collected from University of Port-Harcourt and other local gardens in Port-Harcourt. Water and Methanol extracts were made from the dried leaves of Scoparia dulcis. The aqueous extract of Scoparia dulcis was prepared by soaking 300g of dried powdered samples in 1000mls of distilled water boiled to about 50oC for 24 hours. The solution was filtered using Whatman filter paper N0.42 (125mm). The filtrate was evaporated to dryness using a rotary evaporator. The water extract weighed about 8.67gm which was stored in sterile containers and preserved in the freezer throughout the course of the study.

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Continental J. Pharmacology and Toxicology Research 4 (1): 30 - 34, 2011

The Methanol extract was prepared by soaking 300g of dried powdered sample in 5 litres of 50% methanol for about 12 hours. The solution containing the active ingredient was filtered using Whatman filter paper N0.42 (125mm). Continuous extraction was done for 24 hours. The filtrate was evaporated to dryness using a rotary evaporator. Methanol extract weighing about 17gm was gotten. Chemical tests were carried out on the aqueous and Methanol extracts of the powdered specimens using standard procedures to identify the constituents as described by Sofowara (1993), Trease and Evans (1989) and Harbourne (1973). 30 rats with body weight ranging between 200-250gm were divided into 6 groups of 5 animals each, and 100mg/kg, 200mg/kg, 300mg/kg, 450mg/kg and 600mg/kg of the water extract was given intraperitoneally to rats in groups 1-5 respectively. The sixth group which was the control group was injected with 0.5ml of normal saline. The LD50 was calculated using the arithmetic method of Karber (Adapted by Aliu). The results were compared to that of the study done by Awantang, et al (2004) (7. 47 + 1.19g/kg); vassourinha monograph (1g/kg). The acute toxicity test was also carried out using the intraperitoneal route for the methanol extract by injecting doses of 50mg/kg, 100mg/kg, 200mg/kg, 300mg/kg and 400mg/kg of the methanol extract intraperitoneally to rats in groups 1 5 respectively. The sixth group was injected with 0.5ml of normal saline and used as the control. The number dead in each group was noted. The LD50 of the methanol extract was calculated using the Arithmetic method of Karber. Doses higher than the LD50 were administered for the methanol and water extracts to 4 groups of 5 rats each at doses of 600mg/kg and 800mg/kg for Methanol extract and 800mg/kg, 1000mg/kg for the water extract. The fifth group had 0.5ml normal saline. Some of the animals that had dose of extract higher than the LD50 died. The brain of the dead rats were dissected and sent for histopathological analysis. The control group was sacrificed and the brain dissected and sent for histopathological analysis. The animal Brain were fixed in 10% formalin solution for about 12 hours, and then dehydrated with graded percentage of alcohol -- 50% alcohol for 2 hours, 70% alcohol for 2hours, 95% alcohol overnight and absolute alcohol (2 changes). The tissues were then cleared with pure xylem solution for 2 hours before infiltration took place, and transferred into molten paraffin wax (M.P 54oC-58oC) for 2 hours on hot plate. The embedded tissue blocks were sectioned with a Shaddon AS 325 rotatory microtone and slides were prepared with the sections. The tissues were stained with Ehrlichs haematoxylin and eosin blue using Lillies method (Lillie, 1976). Each of the slides was placed in the photomicroscope and photographed under magnifications of x40; the photographic transparencies were obtained and later developed into photo micrographic pictures which were then scanned, copied and pasted electronically on the result page. RESULTS The results of the various components of the study are as presented below. PHYTOCHEMICAL SCREENING The phytochemical study carried out on the leaves of Scoparia dulcis plant reveals the presence of Tannins, Phlobatannins, Steroids, Terpenoids, Flavonoids and Cardiac glycosides, however it does not contain Saponins. BRAIN In the rats that received 600mg/kg of the methanol extract, the brain tissue revealed features of edema with few lymphocytes seen as is depicted in Figure 2. In the rats that received 800mg/kg water extract, there was dense chronic inflammatory cell infiltrate but no features of edema were seen as shown in Figure 3. In the rats that had 1000mg/kg water extract, the Menninges were preserved, with congestion of vessels, edema and fibrinous deposits and infiltration by lymphocytes as seen in Figure 4.

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Continental J. Pharmacology and Toxicology Research 4 (1): 30 - 34, 2011

Fig. 1 Brain (40). Control group, 0.5ml Normal saline with normal brain architecture.

Fig. 2 Brain (40). 600mg/kg Methanol extract. Brain tissue reveals features of cerebral edema (A) and lymphocytic infiltration(B) within 24 hours.

Fig. 3 Brain (40). 800mg/kg water extract. There is chronic inflammatory cell infiltrate(A) within 24 hours.

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Continental J. Pharmacology and Toxicology Research 4 (1): 30 - 34, 2011

A B

C
Fig. 4 Brain (40). 1000mg/kg water extract. There are congested vessels (A), edema (B) lymphocytic infiltration and fibrinous deposit(C) within 24 hours. DISCUSSION Scoparia dulcis plant contains compounds that have anti-inflammatory properties (Freire et al 1993). This work has shown that Scoparia dulcis extract is a potent pro inflammatory agent when given in high / toxic doses as evidenced by the dense infiltration by inflammatory cells in the brain of the rats that had Scoparia dulcis but not in the control group that had normal saline. Its pro-inflammatory activity is also dose-dependent as the infiltrate increased with increase in the dose of the extract. In concordance with the physical findings on injection of the extracts these reports reveal toxic effect of the drug on the brain which can be safely extrapolated to humans who are more at risk of its toxicity due to its wide usage. In this work, Scoparia dulcis also caused cerebral edema and vascular congestion in the rats that received 600800mg/kg of the methanol extract and 1000mg/kg of the water extract. There was also infiltrates of lymphocytes which became more marked with increasing doses of the extract. This implies that the chemical components of the extract that caused these effects could cross the blood brain barrier. Cerebral edema is classified as: 1. Vasogenic edema The disruption of the cerebral capillary provides the underlying mechanism for vasogenic edema. The amount of edema is greatest in the white matter (increased water and sodium in the extracellular spaces, decreased potassium); but the same changes may take place in grey matter but less so. The astrocytes become swollen. This type of edema is seen in response to trauma, tumors, focal inflammation, and late stages of cerebral ischemia. 2. Cytotoxic edema This is due to the derangement in cellular metabolism resulting in inadequate functioning of the sodium and potassium pump in the glial cell membrane. As a result there is cellular retention of sodium and water. There are swollen astrocytes in grey and white matter. Cytoxotic edema is seen with various intoxications (dinitrophenol, triethyltin, hexachlorophene, isoniazid) and in Reye's syndrome, severe hypothermia, and early ischemia. 3. Osmotic edema Normally CSF and ECF osmolality in the brain is slightly greater than that of plasma. When plasma is diluted by SIADH, water intoxication, hemodialysis, there is passage of water down abnormal gradient creating cerebral edema. 4. Hydrostatic: This form of cerebral edema is seen in acute, malignant hypertension. It is thought to result from direct transmission of pressure to cerebral capillary with transudation of fluid into the ECF. Pathophysiology of Cerebral Edema Pathophysiologically, cerebral edema is a cascade of events involving loss of the integrity of the blood brain barrier (BBB), setting up of a vascular hydrostatic gradient, increased tissue pressure, decreased cerebral blood

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flow, with resulting tissue acidosis. The cerebral capillary is the site of the BBB. Interendothelial tight junctions impede the passage of electron-dense markers. Cerebral capillaries lack fenestrations and have tight junctions. They are active in the process of pinocytosis. All these features regulate the passage of highly polar hydrophilic molecules allowing virtually unrestricted passage of lipid-soluble substances. Cerebral capillaries have 2-4 times the number of mitochondria as other capillaries. Cerebral capillaries have an array of important enzymes: ATPase, dehydrogenase, monamine oxidase, DOPA decarboxylase, acid and alkaline phosphatase, NAD and others. BBB has both central (cholinergic and aminergic) and peripheral (sympathetic and parasympathetic) innervations. However some areas are without the BBB are the choroid plexus, area postrema, median eminence, neurohypophysis, pineal body, subforniceal organ, commissural organ, supra-optic crest (Harrigan, 1996). Extracts of Scoparia dulcis are highly toxic to the brain of rats. These findings are important in that, though Scoparia dulcis is a widely used medicinal plant, with very scanty works published on its toxicity, it has the potential for life threatening toxicity when used in high doses. Both extracts has potent pro inflammatory effect and tendencies to cause cerebral edema. This implies that this plant could be a good pharmacologic tool in the experimental induction of systemic inflammatory reaction and cerebral edema hence ultimately in the screening of drugs that could be used in the treatment of these conditions. Also in clinical cases of toxicity where it is confirmed that the offending plant is Scoparia dulcis, treatment should be directed at management of cerebral edema as this is one of the most affected organs in the body. The indiscriminate use of herbal plants is not without its adverse effects. There had been many unreported cases of toxicity and adverse effects following use of medicinal plants resulting in organ damage and even death. In view of this, there should be continuous monitoring of the safety of herbal products. The principles of pharmacovigilance should be applied in the monitoring of use of this herbal product as this will help in documenting exposure and toxicity from this medicinal plant. REFERENCES Freire SM, Torres LM, Souccar C, Lapa AJ. (1993). Analgesic and anti-inflammatory properties of Scoparia dulcis L. Extract and Glutinol in rodents. Phytother Res. 7 6:408-14. Harborne J.B (1973). Phytochemical methods, London. Chapman and Hall, Ltd. Pg. 149 88. Harrigan MR. (1996). Cerebral salt wasting syndrome. Neurosurgery 38: 152-60. Lillie R.D, Fullmer H.M. (1976). Histopathologic Technique and practical Histochemistry. 4th Edition. NY . MacGrawhill. pg. 88-92. Okwu DE (1999). Flavouring properties of spices on cassava Fufu. Afr.J. Roots Tuber Crops 3(2): 19-21. Okwu DE (2001). Evaluation of the chemical composition of indigenous spices and flavouring Agents. Global J. Pure Appl. Sci. 7(3): 455-459. Orhue, N.E.J. and Nwanze, E.A.C. (2004). Effects of Scoparia dulcis on Trypanosoma brucei Induced Alterations in serum Transaminase, Alkaline Phosphatase and Bilirubin in the Rabbit. J. Med. Sci., 4 (3): 194-7. Sofowara A (1993). Medicinal plants and traditional medicine in Africa. Ibadan, Nigeria. Spectrum Books Ltd, Pg: 289. Trease, G.E, Evan W.C. (1989). Pharmacognosy. 11th edition. Brailliar Tridel Can. Macmillan publishers. Received for Publication: 26/06/11 Accepted for Publication: 26/08/11 Corresponding author Esume C.O Department of Pharmacology, Delta State University Abraka and Department of Medicine, Central Hospital, Warri

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