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2 Analgesic Activity of Ethanol Leaf Extract Of Danraka et al., Nig. Journ. Pharm. Sci., September, 2020, Vol. 19 No.2, 11-24 Nigerian Journal of Pharmaceutical Sciences Vol. 19, No2, 2020, ISSN: 0189-823X 2 All Rights Reserved ANALGESIC ACTIVITY OF ETHANOL LEAF EXTRACT OF HYMENODICTYON FLORIBUNDUM (Hochst. & Steud) B. L. Rob AND IT’S POSSIBLE MECHANISM OF ACTION IN MICE *Danraka, R.N., Maje, I. M. and Ejiofor, J. I Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. *Author for correspondence: [email protected] ABSTRACT The plant Hymenodictyon floribundum is a common shrub in Africa that has been reported to be used traditionally in the treatment of pain and inflammatory diseases. In this study, the analgesic activity of the ethanol leaf extract of Hymenodictyon floribundum (HF) in mice was studied after phytochemical screening and LD50 determination. HF (375, 750 and 1500 mg/kg p.o) was evaluated for analgesic activity using acetic acid induced writhing and hot plate methods in mice. The ethanol leaf extract exhibited significant (P < 0.05) and dose-dependent analgesic activity comparable to that of the reference drug morphine (10 mg/kg body weight, p.o) in both acetic acid and hot plate method. The preliminary phytochemical screening of the extract revealed the presence of flavonoids, tannins, saponins, steroids glycosides, anthraquinones and phenols. The oral median lethal dose (LD50) of the extract in mice was found to be greater than 5,000 mg/kg body weight. The pretreatment of mice with prazosin, naloxone, and propranolol significantly (p<0.05) decreased the analgesic effect of the extract, while pretreatment with yohimbine, and glibenclamide, each, had no significant effect on its analgesic activity. The findings suggest that the ethanol extract of HF possesses analgesic activity possibly via opioidergic, α1 and β-adrenergic receptors pathways and supports the ethnomedicinal claims of the plant for the management of pain. Keywords: Hymenodictyon floribundum, analgesic, phytochemical, writhes, acetic acid, hot plate INTRODUCTION The International Association for the Study of Pain and the World Health Organization Research in phytomedicine is now a define pain as “an unpleasant sensory and common practice in the search for bioactive emotional experience associated with actual substances used as medicines (Sen and or potential tissue damage, or described in Samantha, 2014). The plant Hymenodictyon terms of such damage.” (IASP, 2014.). floribundum belongs to the family of Although the drugs used in the treatment of Rubiaceae with a grey-black stem that is 4 to pain are available, people still complain of 9 meters tall and grows in a rocky terrain. pain. Under-treated or unrelieved pain also (Bridson and Verdcout, 2003). There are places a high financial burden on humans over 30 species of Hymenodictyon and (Stewart et al 2003). Most of the available several ethnobotanical claims have pointed analgesic like the non-steroidal anti- to the use of H. floribundum as an effective inflammatory drugs (NSAIDs) cause anti-inflammatory and analgesic plants. H. gastrointestinal bleeding and excelsum for example has been scientifically immunosuppression (Hougee, 2008), while proven to possess significant anti- the relatively safe cyclooxygenase-inhibitors inflammatory and analgesic properties (Kar such as celecoxib, are often not affordable to et al., 2013). the general populace (Tick et al.,2018). In a 11 research sponsored by World Health floribundum were shade-dried until constant Organization to study the global burden of weight was obtained and then reduced into disease, it was found that conditions fine powder with the aid of a mortar and characterized by presence of pain (low back pestle. The powdered leaf material (700 g) pain, neck pain, other musculoskeletal was extracted exhaustively with aqueous w disorders, migraine, and falls) accounted for ethanol (70% /v) using continuous Soxhlet 5 of the top 10 conditions. (Vos et al., apparatus for 72 hours. The extract was 2017). Low back pain was responsible for concentrated to dryness on a water-bath set 83 million disability and was the greatest at 50°C, subsequently packed in an amber contributor of years lived with disability bottle and stored in a desiccator prior until (Hartvigsen et al., 2018). Since traditional use. The extract was referred to as ethanol medicine has been the major source of most leaf extract of Hymenodictyon floribundum drugs and has better acceptability because of (EEHF). Solutions of the extract were perceived safety (wang et al.,2014), it safe prepared freshly with Normal Saline for to say that there is a possibility of more each study. The percentage yield of the discovery through traditional medicine. extract was calculated using the formula: Though not documented, the leaves of Hymenodictyon floribundum like H. excelsum, is claimed to possess analgesic and anti-inflammatory properties by the Qualitative Phytochemical Analysis ethnic people of kuregu, Zaria Local Government Area, Kaduna state and hence The extract was subjected to phytochemical the need to validate this claim screening in accordance with standard protocol as described by Evans (2002). The 2.0 MATERIALS AND METHODS extract was screened by adopting simple chemical tests to detect the presence of Plant Collection and Identification alkaloids, flavonoids, saponins, cardiac glycosides, tannins, anthraquinones, The whole plant consisting of the stem (with carbohydrates, steroids and triterpenes. the leaves attached) and the root was collected from Kargi Hill along Birnin Animals Gwari road, Zaria Local Government Area, Kaduna state in October 2019. The collected Adult male Swiss Albino mice (18-25g) plant was identified and authenticated, by were obtained from the Animal House comparing with an existing specimen by a Facility of the Department of Pharmacology taxonomist, Malam Sanusi Namadi of the and Therapeutics, Ahmadu Bello University, Department of Botany, Faculty of Life Zaria, Nigeria. The animals were housed in Sciences, Ahmadu Bello University, Zaria. a well-ventilated room in their cages and A voucher specimen number of ABU900124 provided with normal rodent feed and water was assigned to the plant by comparing with ad libitum until the end of the study. They an existing voucher specimen. were maintained under normal 12 hours light/dark cycle. All experiments were Extraction of plant material conducted in accordance with the guideline of National Institute of Health (NIH, 1998). Plant extraction was carried out according to the method described by Kupchan et al. Ethical approval for the use of animals was (1973). The leaves of Hymenodictyon sought from the Ahmadu Bello University 12 Committee on Animal Use and Care At the end of this test, mice that survived (ABUCAUC) and an approval number of were euthanized with chloroform and ABUCAUC/2020/011 was given. subsequently incinerated 2.4 Drugs/chemicals Evaluation of Analgesic Activities in Mice Glacial acetic acid (May and Baker limited, Acetic acid induced writhing in mice Dagenham, England), ethanol (Sigma Aldrich, St. Louis Mo, USA), morphine The method previously described by Koster sulphate (Martindale Pharmaceuticals, UK), et al (1959) was adopted for this study. hydrochloric acid (May and Baker, UK), Thirty (30) male Swiss albino mice were sulphuric acid (May and Baker, UK), ferric divided into 5 groups of six (6) mice. Mice chloride anhydrous (Avishkar, India), in group I were administered with Normal ammonia (Lobachemie, India), naloxone Saline (10 ml/kg, p.o) and served as a hydrochloride (Abcam Plc, Cambridge, negative control, while mice in group V UK), prazosin hydrochloride (Abcam Plc, received morphine (10 mg/kg, p.o.) and Cambridge, UK), glibenclamide (Abcam served as a positive control. Group II, III Plc, Cambridge, UK), yohimbine and IV received graded doses of EEHF (375, hydrochloride (Abcam Plc, Cambridge, 750 and 1500 mg/kg respectively) via oral UK), propranolol hydrochloride (Abcam Plc, route. Sixty (60) minutes after oral Cambridge, UK). All the reagents were of administration, acetic acid 0.6% v/v (10 analytical grade. ml/kg) was administered to each mouse via intraperitoneal route and was placed in an Acute Toxicity Studies observation cage. Five (5) minutes after acetic acid injection, the number of writhes Median Lethal Dose (LD50) determination was counted for each mouse for a period of was conducted using Organization for 10 minutes. A reduction in the number of Economic Co-operation and Development writhes as compared to the vehicle treated (OECD 423) guidelines in mice. In this animals was considered as evidence for the method, two groups of three (3) animals presence of analgesia and expressed as each were fasted prior to dosing (food but percent inhibition of writhes. not water was withheld for 3 hours). The fasted body weight was determined for each animal and the dose was then calculated according to the body weight. Food was further withheld for 1-2 hours in mice after Mc = Mean number of writhes (control) the extract has been administered. In the first Mt = Mean number of writhes (test) phase, 2000 mg/kg of the test substance was administered to each mouse in a single oral Hot Plate test in Mice dose using oral canula and the mice were observed for 48 hours for clinical sign and The method of Eddy and Leimbach (1953) symptoms of toxicity including death. The was adopted. The hot plate was provided by second phase was conducted in a similar Ugo Basile hot plate (model 7280, manner to the first but with dose of 5000 Germany) at the laboratory of the mg/kg. Department of Pharmacology, Faculty of Pharmacy, Bayero University Kano, Nigeria. Mice were individually placed on a 13 hot plate (55±1°C), before drug treatment so various pain pathways involved in the that each animal serves as its own control. analgesic activity of H. floribundum. The The time taken for the animal to either pathways investigated and the standard licked the paw, withdrawal of the paws or antagonists/blockers used for the studies jumped was taken as reaction time and were as follows: recorded.
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