ANTINOCICEPTIVE PROPERTY OF Costus afer Ker STEM JUICE AND ETHANOL LEAF EXTRACT IN ALBINO RATS

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1 Comprehensive Journal of Medical Sciences Vol. 2(2), pp , August ISSN Copyright 2014 Knowledgebase Publishers. RESEARCH ARTICLE ANTINOCICEPTIVE PROPERTY OF Costus afer Ker STEM JUICE AND ETHANOL LEAF EXTRACT IN ALBINO RATS Ijioma, S.N. 1, Nwosu, C.O. 1, Emelike, C.U. 2, Okafor, A.I. 3, Nwankwo, A.A. 4 Department of veterinary Physiology, Pharmacology, Biochemistry and Animal Health, College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, Nigeria. 1 Diagnostic Laboratory Unit, University Health Services, Michael Okpara University of Agriculture, Umudike, Nigeria. 2 Department of Animal and Environmental Biology, Abia State University, Uturu, Nigeria. 3 Department of Human Physiology, Abia State University, Uturu. 4 Corresponding Author nedumeme@gmail.com Accepted 12 th August, 2014 This study investigated the antinociceptive property of ethanol leaf extract of Costus afer ethanol leaf extract (CALE) and the stem juice (CASJ) in rats, using different rat models - acetic acid, tail flick and tail immersion assays. Forty albino Rats were used for each experimental model. Varying doses of CALE: 100, 200, and 400mg/kg and CASJ: 2.5, 5, and 10ml/kg were administered orally to rats in different groups. The negative control group received 0.2ml normal saline while Aspirin (20mg/kg) was used as reference drug. Results obtained showed that both CALE and CASJ exerted a dose dependent reduction in number of writhes per time as 400mg/kg CALE and 10ml/kg CASJ inhibited the number of writhes observed in the negative control group by 75.38% and56.38% in the acetic acid model respectively, 177.4% and % in the tail flick model and and % in the tail immersion assay respectively. The effects of CALE and CASJ were significantly (P<0.05) different from the negative control group and compared favorably with the effects of Aspirin. The experiments therefore indicate that CALE and CASJ could contain principles with strong antinociceptive properties and raises in the search for new antinociceptive agents with minimal side effects and high potency for the management of pain related body diseases. Key Words: Antinociception, Aspirin, Assay, Costus afer, Cyclo-oxygenase-2, Pain, Prostaglandin INTRODUCTION Many plants are today being scientifically studied for bioactive substances and for medicinal potentials, Costus afer appears to be among the least evaluated and as such has scanty published literature on its physiological and pharmacological dynamics. The plant is commonly called Ginger lily or Bush cane in

2 15. Ijioma et al English, Kaki zuwa by the Hausas, Okpete or Okpoto by the Igbo s and TeteOgun by the Yoruba s, all of Nigeria is (Ukpabi et al., 2012; Anaga et al., 2004), is a tall perennial semi-woody herb with leafy canes which may grow up to 3m high and belonging to family Costaceae (Bukil, 1985). Costus afer bears terminal inflorescence of white and yellow flowers and is commonly found in the forest zones of most places including Senegal, Nigeria, South Africa, Guinea, Ghana, and Cameroun and in most regions in tropical Africa, particularly in higher rainfall areas (Bukil, 1985; Ukpabi et al., 2012). Phytochemical analysis of the leaf reveal the presence of alkaloids, saponins, tannins, flavonoids, phenols, glycosides and terpenoids (Momoh et al., 2011; Ukpabiet al., Anaga et al., 2004). The root extract is reported to be used as genital stimulant, laxative and treatment of leprosy and stomach troubles, while the stem juice/sap has been used to treat arthritis rheumatism and pharyngeal infections (Bukil, 1985). The hypoglycaemic property of ethanol leaf extract of Costus afer have been reported (Momoh et al., 2011), while Ukpabi et al.,(2012) reported its ability to alleviate carbon tetrachloride induced hepatic oxidative stress and toxicity. Antinociceptic agents are drugs used to remove/ relief the sense of pain without loss of consciousness. These agents therefore provide temporary relief from pain and may act centrally by reducing the flow of pain signals from the brain, e.g opium, morphine, heroine, codeine(john et al., 1999)or may act locally at the actual site of pain to reduce the amount of pain causing chemicals produced e.g Ibuprofen, Diclofenac Sodium, Aspirin, and lots more. This study was designed to evaluate the antinociceptive properties of Costus afer ethanol leaf extract (CALE) and stem juice (CASJ) using three different animal experimental models with a view to verify/validate the use of the extracts in traditional medicine for the treatment of pyrexia, rheumatism and arthritic pains and to improve on the scanty published literature available on the plant. 2.0 MATERIALS AND METHODS Collection of Plant and Preparation of Leaf Extract (CALE) Fresh leaves of Costus afer were collected from a bush in Owerri-Aba, Ugwunagbo Local Government Area of Abia State, Nigeria. The extract was prepared using a modified method of Akah et al.,(2009). The collected leaves were air dried at room temperature for 14 days after which they were ground to coarse powder using a manual blender. Thirty five (35) grams of the powdered material was introduced into the extraction chamber of the soxhlet extractor and extraction was done using ethanol as solvent. Extraction temperature was maintained at 70 0 C for 48hours. At the end of the period, the ethanol was evaporated at low temperature in an electric oven to obtain a crude extract which weighed 4.85g and represented a yield of 13.86% Extraction of Costus afer Stem Juice (CASJ) The collected stems were debarked after that the foliage leaves covering them were removed. The debarked stems were then introduced into a clean manual blender and were crushed to squeeze out their juice. The resulting juice was filtered using a filter paper to obtain about 20ml of the filtered juice. 2.2 Animals A total of 30 Mice (25 30g) and 120 Rats (95-130g) obtained from the Animal Production unit of the College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, were used for the study. They animals were fed with standard pelleted feed (Vital Feed, Nigeria), with water ad libitum, but starved for 12 hours prior to commencement of Experiment. All Animal experiments were conducted in compliance with NIH guidelines for care and use of Laboratory Animals (Pub. No , Revised 1985), as expressed by Akah et al., (2009). The study was carried out in the Physiology Laboratory of the Department of Physiology, Pharmacology, Biochemistry and Animal Health, Michael Okpara University of Agriculture, Umudike, Nigeria.

3 Ijioma et al Experiments Acute toxicity study Thirty mice of both sexes were divided into 6 groups of 5 mice each and were assigned graded oral doses of Costus afer ethanol leaf extract in the order 500, 1000, 2000, 3000, 4000 and 5000mg/kg body weight. After administration, the animals were allowed free access to feed and water and the number of deaths in each group was noted at the end of 24 hours. LD50 was to be calculated using Karber s formular as expressed Enegide et al., (2013). LD50 = LD100 - ( Dd x Md) N Where: LD50 = Dose that killed 50% of animals in a group LD100 = Dose that killed all animals in a group (Dd x Md) = Summation of all products of dose difference and mean deaths, and N = Number of animals in each group Effects of CALE and CASJ on Acetic Acid Induced Writhing Test A modified method of Anaga et al., (2010) was employed. Forty rats were divided into 8 groups of 5 rats each. Group 1 was given 0.2ml normal saline and served as the negative control. Group 2 received Aspirin (20mg/kg). Groups 3, 4, and 5 were treated with 100, 200, and 400mg/kg body weight of CALE, while groups 6, 7, and 8 received 2.5, 5 and 10ml/kg body of CASJ. All treatments were done by the oral route. 30 minutes after treatment, each rat was given an intraperitoneal (I.P) injection of 0.6% acetic acid at a dose of 10ml/kg body weight. The number of writhes (stretching of hind limbs and bending of trunk) made by each rat was counted for 20 minutes. Percentage inhibition was evaluated using the expression: Percentage Inhibition = Writhes in test Writhes in control x 100 Writhes in control Tail Flick Assay A modification of the method of Esam, (2011), was employed. Forty rats were divided into 8 groups of 5 rats each. Group 1 was given 0.2ml normal saline and served as the negative control. Group 2 received Aspirin (20mg/kg). Groups 3, 4, and 5 were treated with 100, 200, and 400mg/kg body weight of CALE, while groups 6, 7 and 8 received 2.5, 5 and 10ml/kg body weight of CASJ. All treatments were administered orally. After 30 minutes of treatment, the lower halves of the tails of the animals were individually dipped into a beaker of cold water (0.1 0 C). The time taken for tail withdrawal from the water was recorded as the reaction time Tail Immersion Test A modification of the method of Esam, (2011), was employed. Forty rats were divided into 8 groups of 5 rats each. Group 1 was given 0.2ml normal saline and served as the negative control. Group 2 received Aspirin (20mg/kg).Groups 3, 4, and 5 were treated with 100, 200, and 400mg/kg body weight of CALE, while groups 6, 7, and 8 received 2.5, 5 and 10ml/kg body weight of CASJ. After 30 minutes of treatment, the lower 5cm portion of each animal s tail immersed in a water bath maintained at 55 0 C. The time taken for tail withdrawal from the water was recorded as the reaction time. Inhibition time ratios were evaluated for the tail flick assay and tail immersion test using the expression: Percentage Inhibition= R2 - R1 X100 R1 1 Where:

4 17. Ijioma et al R1 = Reaction time in negative control and R2 = Reaction time in test Statistical Analysis All data were expressed as mean+ Standard Error of Mean (SEM) and analyzed using student s t-test. P values less than 0.05 at 95% level of significance for tests versus control were adjudged significant. 3.0 RESULTS 3.1 Acute toxicity (LD50) During the period of acute toxicity, all the mice administered CALE had normal disposition, were active and survived the 24 hours period of study with no signs of toxicity, even at an oral dose of 5000mg/kg body weight. 3.2 Effect of CALE and CASJ on Acetic Acid Induced Writhing Reflexes in Rats All doses of CALE and CASJ significantly (P<0.05) decreased the mean number of writhes in the treated rats when compared to the negative control, with maximum inhibitions of 75.38% and 56.03% achieved in groups administered CALE (400mg/kg) and CASJ (10ml/kg) respectively. These dose dependent effects of CALE and CASJ the compared favorably with that of the reference drug used (Table 1). Table 1: Effects of CALE and CASJ on acetic acid induced writhing in rats Group Treatment Number of writhes Per 20 % Inhibition Minutes ml normal saline mg/kg, Aspirin * mg/kg, CALE * mg/kg CALE * mg/kg CALE * ml/kg CASJ * ml/kg CASJ * ml/kg CASJ 31.20± *P< 0.05 when compared to negative control group 3.2 Effects of CALE and CASJ on reaction time in tail flick and tail immersion assays in rats. All doses of the extract significantly (P<0.05) increased the reaction times in tail flick and with 400mg/kg of CALE and 10ml/kg of CASJ achieving maximum inhibitions of 177.4% and % respectively in the tail flick assay (Table 2) while the doses achieved maximum inhibitions of 260.6% and % in the tail immersion assay (Table 3). The effects were dose dependent and also compared favorably with that of Aspirin.

5 Ijioma et al. 18 Table 2: Effects CALE and CASJ on reaction time in the tail flick assay Group Treatment Reaction time in seconds %Inhibition ml normal saline mg/kg, Aspirin * mg/kg CALE * mg/kg CALE * mg/kg CALE * ml/kg CASJ * ml/kg CASJ * ml/kg CASJ 79.05± *P<0.05 when compared to negative control group Table 3: Effect of CALE and CASJ on reaction time in the tail immersion assay Group Treatment Reaction time in seconds %Inhibition R ml normal saline mg/kg, Aspirin * mg/kg CALE * mg/kg CALE * mg/kg CALE * ml/kg CASJ * ml/kg CASJ * mg/kg CASJ 69.11± P<0.05 when compared to negative control group. DISCUSSION Results obtained indicate that the ethanol leaf extract of Costus afer (CALE) and Costus afer stem juice (CASJ) are safe and could be well tolerated after oral consumption since no death was recorded even at a high dose of 5000mg/kg body weight. Both CALE and CASJ reduced the number of writhes and increased reaction time in the experimental rats exposed to pain stimuli via the different models - acetic acid induced writhing, the tail flick assay and the tail immersion assay. The results suggest that both CALE and CASJ contain active principles with analgesic properties. Momoh et al., (2011) had reported that Costus afer contain phytochemical substances including alkaloids, flavonoids, tannins, phenols, glycosides and terpenoids, some of which have been implicated in antinociception. CALE and CASJ may have achieved these antinociceptive effects by inhibiting the activity of cyclo-oxygenase-2 (cox-2), causing the stoppage of prostaglandins formation. Prostaglandins are chemical substances which stimulate the noci receptors in the body leading to the sensation of pain (John et al., 1999).The extract may also have interfered with G-protein mediated signal transduction, an analgesic mechanism unrelated to inhibition of prostaglandin synthesis. It also may have augmented the peripheral mechanism through interference with the formation of prostaglandins in the central nervous system. These mechanisms have been implicated in the forms of analgesia induced by non-steroidal anti- inflammatory drug (NSAID), such as Aspirin (Cashman, 1996; Steve and Renne, 2014; Vane and Botting, 2003). The result of this current study tends to agree with the traditional use of Costus afer extract in the management of Arthritis and for the relief of pain and inflammations as reported by Bukil, (1985). In conclusion, these results suggest that ethanol leaf extract and stem juice of Costus afer plant are safe and potent agents with strong antinociceptive properties and may be of value in the management of all forms of Arthritis, Rheumatism and other conditions associated with pain. With the gastrointestinal toxicity associated with the use of Aspirin and other orthodox analgesics, Costus afer leaf extract and stem juice could be used as a main therapy in pain management.

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