ORIGINAL PAPER https://dx.doi.org/10.4314/njpr.v16i2.4S Nig. J. Pharm. Res. 2020, S1 pp 31-37 ISSN 0189-8434 e-ISSN 2635-3555 Available online at http://www.nigjpharmres.com Studies on Analgesic and Anti-Inflammatory Activities of Aerial Parts of Tephrosia Bracteolata GUILL. and PERR. (Fabaceae) in Rodents A. A. SADAM*1ABCDE, S. M. ABDULLAHI1AEF., U. U. PATEH, 1AF, A. SHEHU2CE, L.O. BAKARE1B 1 Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria, Nigeria. 2 Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation; D – writing the article; E – critical revision of the article; F – final approval of article. Abstract Background:Tephrosia bracteolata is a widespread shrub belonging to the family (Fabaceae) and genus Tephrosia. It is traditionally used for treating rheumatic pains, dropsy and stomach ache. Objectives:In view of the ethnomedicinal claim and the continuous search for new medicinal agents, the phytochemical constituents, analgesic and anti-inflammatory activities of chloroform fraction (CF) of the methanol extract of Tephrosia bracteolata in mice and rats was evaluated. Methods:Preliminary phytochemical screening was conducted using standard method. Analgesic activity of CF (100, 200 and 400 mg/kg body weight orally) was investigated using acetic acid-induced writhing test and thermally induced pain model in mice. Additionally, anti-inflammatory activity was tested by carragenaan-induced paw edema in rats. Results:Phytochemical screening revealed the presence of alkaloids, triterpenes and flavonoids. The oral LD 50 of CF was above 2000 mg/kg body weight. CF significantly (p<0.05) and dose dependently reduced the number of writhes with percentage inhibition of 47.76 48.41 and 72.6 % at dose of 100, 200 and 400mg/kg respectively. CF also significantly (p<0.05) and dose dependently increased the mean reaction time. At dose of 400 mg/kg, CF at 60 and 90 minutes exhibited greater activity when compared to the standard agent pentazocine. CF(200 and 400 mg/kg) at times 3, 4 and 5 hours significantly (p<0.05) decreased the paw edema in rats when compare with the ibuprofen treated group. Conclusions:The chloroform fraction of the methanol crude extract of Tephrosia bracteolata possesses analgesic and anti-inflammatory activities. Keywords: Tephrosia bracteolata, Phytochemical screening, Toxicity, Analgesic activity, Anti-inflammatory activity INTRODUCTION Medicinal plants have been estimated to be the primary source of health care needs of over 80% of the world population living in developing countries (Gabriel et al., 2018). The process of searching useful plants from different records to the development of methods for the industrial production of drugs is termed Ethnopharmacology and it plays an important role in the discovery of new biologically active compounds (Dong., 2013). Tephrosia bracteolata is a shrub of widespread belonging to the family Fabaceae that grows in uncultivated areas of tropical and warm-temperate regions. There are approximately 400 species included in this genus (Burkill., 1985). Onaolapo et al., 2004 reported the toxicity and anti-pyretic studies of the crude methanolic extract of Tephrosia bracteolata leaves, the study demonstrated that the 31 Sadam et al./Nig.J.Pharm. Res. 2020, S1:31-37 plant possess potent anti-pyretic activity. Egharevba et al., 2020 reported the antidiabetic, antioxidant and antimicrobial activities of extracts of Tephrosia bracteolata leaves. Other biological activities reported on other genus of the plant include anticancer (Hassan et al., 2017) and anti-plasmodial activities (Nondo et al., 2014). Tephrosia genus have been reported to possess several phytoconstituents that can be related to various biological activities including Isopongaflavone from Tephrosia bracteolata (Khalid et al., 1981), obovatin methyl ether from Tephrosia aequilata (Atilaw et al., 2017), kaempferol 3-O-β-D-glucopyranoside from Tephrosia calophylla (Ganapaty et al., 2009). Pain is an ill-defined, unpleasant sensation, usually evoked by an external and internal noxious stimulus linked to tissue damage (Chatterjee et al., 2015). It is physiologically associated with receptors, confirmed by electrophysiology methods in which the intensity is dependent on internal or external factors and ends up in the brain (Yam et al., 2018). Additionally, pain may also be generated from peripheral and central METHODOLOGY Materials Drugs and reagents Analytical grade chemicals and reagents were used including; n-hexane, chloroform, ethyl acetate, methanol, acetic acid, sulphuric acid (Sigma-Aldrich, USA), distilled water and tween 80, Molisch’s reagent, Dragendorff’s reagent, Mayer’s reagent and Shinoda’s reagent, carrageenan (Sigma Aldrich), Acetic acid (Searle Essex, England), ibuprofen (Lek, Slovenia), pentazocine (Martinadale, Essex) and Piroxicam (Pfizer laboratories). Experimental animals Swiss albino mice (20-38 g) and wister rats of either sex (100-162 g) were obtained from the Animal House Facility of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University Zaria, Nigeria. They were maintained under standard conditions in propylene cages, fed with laboratory diet and water ad libitum. Methods Plant collection and identification The aerial parts of the plant (T. bracteolata) was collected from bushy village of Samaru, Sabon gari Local Government, Kaduna State, Nigeria in October, 2018. The plant was authenticated at the herbarium unit, department of botany, Ahmadu Bello nervous system (Grichnik et al., 1991). Inflammation is a complex biological response of the body tissues to harmful stimuli like pathogens, damaged cell or irritants (Ferrero-Miliani et al., 2007). The classical known symptoms of inflammation include redness, pain, swelling and heat (Medzhitov., 2008). Pain and inflammation can be classified as acute and chronic. Analgesics are drugs that selectively relieve pain by acting on the CNS or peripheral pain mechanisms, without significantly altering consciousness (Tripathi., 2003). These drugs have serious limitation due to their side effects such as gastrointestinal irritation/ulceration, safety, tolerance, dependency and respiratory depression (Howland and Mycek., 2006). Due to these drawbacks, it is necessary to conceptualize search for newer potent analgesic and anti-inflammatory agents with better efficacy, lesser side effects, easily available and accessible. This study aimed to establish the analgesic and antiinflammatory effect of the aerial part of Tephrosia bracteolata University, Zaria by comparing with voucher specimen number (0385). The aerial part was shade dried, pounded to powder and stored at room temperature for use. Extraction and Partitioning The pulverized plant material (2 kg) was extracted exhaustively with methanol using maceration method for four days with occasional shaking. The extract was concentrated in-vacuo using rotary evaporator at 40 0C which afforded 190 g greenish-brown crude methanol extract (CME). Thereafter 150 g of CME was partitioned with hexane, chloroform and ethyl acetate in order of polarity to obtain the residual fractions. The chloroform fraction was utilized for further study Phytochemical screening Preliminary phytochemical screening of the CF extract was investigated for the presence of alkaloids, steroids, triterpenes, flavonoids, tannins, glycosides and flavonoids using standard methods (Trease and Evans., 1996). Oral acute toxicity profile The median lethal dose (LD50) of the CF extract was conducted according to OECD method (2000) in mice and rats. In the first phase, 3 animals were administered the extract with dose 5000 mg/kg body weight orally. The animals were observed for mortality within a period of 2 weeks. Thereafter. In 32 Sadam et al./Nig.J.Pharm. Res. 2020, S1:31-37 the second phase, a limit test at one dose level of 2000 mg/kg body weight of the extract is carried out with 3 animals and observed for signs and symptoms of toxicity and mortality as stated above. LD50 was calculated using the formula. LD50 = 20% 𝑜𝑓 𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑡𝑜𝑙𝑒𝑟𝑎𝑡𝑒𝑑 𝑑𝑜𝑠𝑒 Antinociceptive Studies Acetic acid induced-writhing test in mice It was conducted according to a method described by Koster et al (1959). Thirty (30) albino mice of either % inhibition = sex were randomly divided into five (5) groups of 6 mice each. Group 1 was administered distilled water 10 ml/kg. Groups 2, 3 and 4 received 100, 200 and 400 mg/kg of the CF extract orally respectively, while group 5 received piroxicam 10 mg/kg body weight orally. An hour later, mice in all groups were administered 10 ml/kg of acetic acid (0.6 % v/v) i.p. Five minutes latency period was allowed and the mice were observed for abdominal writhes. The number of abdominal writhes was counted for a period of 10 minutes. Percentage inhibition of writhes was determined using equation 1 Mean number of writhes (control)– Mean number of writhes (test group) Mean number of writhes (control) Thermal Induced Pain in Mice The test was conducted according to the method described by Eddy & Leimbach (1953). Thirty (30) mice were divided into 5 groups of 6 mice each. The first group received distilled water as the negative control. Groups 2, 3 and 4 were administered with 100, 200 and 400 mg/kg body weight of the CF orally respectively, while group 5 received 10 mg/kg pentazocine i.p. An hour later, each mouse was placed on hot plate (Gallenkamp thermostat) kept at 45±2 ˚C. Reaction time was monitored with stop watch. Index of pain response latency was calculated as the time mouse licked its paw or jump up from the hot plate at time intervals of 30, 60 and 90 minutes after administration of drug. Baseline reaction time for each animal was recorded at zero time reading and a cut off period of 15s was observed to avoid damage to the paw. Anti-inflammatory studies Carragenan-induced paw oedema Anti-inflammatory study was carried out according to the method described by Winter et al. (1962). RESULTS Preliminary phytochemical screening: Phytochemical constituents found present in CF were alkaloids, steroids, triterpenes and flavonoids Acute toxicity study (LD50) The oral median lethal dose (LD50) value for CF was estimated to be greater than 2000 mg/kg body weight. × 100 ……………. equation 1 Twenty-five (25) rats were divided into 5 groups of 5 rats each. Group 1 received 10 ml/kg of distilled water orally. Groups 2, 3 and 4 were administered 100, 200 and 400 mg/kg of CF orally respectively and group 5 received 10 mg/kg of ibuprofen orally. Thirty minutes later, each rat was injected with 0.01 mL of 1 % v/v solution of carrageenan in the sub plantar region of the left hind paw. The paw diameter (cm) was taken at time zero prior to treatment and subsequently measured at time 1, 2, 3, 4, 5 hours after injection of carrageenan using vernier caliper. Statistical Analysis The data obtained for the acetic acid-induced writhing test in mice was subjected to one way analysis of variance (ANOVA) followed by Dunnett’s Post Hoc test for multiple comparisons. Also the data obtained for the thermally induce hyperalgesia and carragenaan-induced paw edema were analyzed using repeated measures ANOVA followed by Bonferoni post hoc test for multiple comparison. Results were considered significant at P≤0.05 and data was expressed as Mean±SEM. Acetic Acid Induced Writhing Test The CF of the methanol aerial extract of T. bracteolata significantly (P <0.05) attenuated the acetic acid-induced abdominal writhes in mice dose dependently. The fraction CF at dose of 400mg/kg gave highest inhibition of abdominal constriction. The standard drug piroxicam at dose of 10 mg/kg significantly (P<0.05) inhibited the acetic acidinduced writhes (Table I). 33 Sadam et al./Nig.J.Pharm. Res. 2020, S1:31-37 Table I: Effect of CF on Acetic Acid Induced Writhing Test in Mice Treatment Dose Mean no. of writhes (mg/kg) ± SEM DW (10 ml/kg) 26.17 ± 5.55 % inhibition 0 CF 100 13.67 ± 3.24* 47.76 CF 200 13.50 ± 1.23* 48.41 CF 400 7.17 ± 1.28* 72.6 Piroxicam 10 4.33 ± 1.33* 83.4 Values presented as Mean ± SEM. Data analyzed using one-way ANOVA followed by Dunett’s PostHoc test. *Significant at p<0.05when compared with the DW group. n = 6. CF = Chloroform fraction of the T.bracteolata, DW= Distilled water Thermal-Induced Pain Test in Mice CF significantly (p<0.05 and dose dependently increased the mean reaction time at dose of 400 mg/kg, at time 60- and 90-minute post treatment (Table II). Table II: Effect of CF on Thermal-Induced Pain Test in Mice (Hot Plate) Treatment Dose (mg/kg) Mean reaction Time (Seconds) at various time 0mins 30mins 60mins 90mins DW 10 ml/kg 3.91±0.53 2.33±0.56 1.92±0.37 1.00±0.00 CF 100 3.16±0.32 7.08±1.07*# CF 200 4.53±0.59 6.45±0.68 7.02±0.83 CF 400 5.96±0.52 7.59±1.12 9.35±1.01 *# 12.36±3.67*# Penta 10 3.77±0.43 7.91±0.79* 8.47±0.47* 6.87±0.99 6.68±1.05 6.50±1.14 7.61±0.79* Values presented as Mean ± SEM. Data analysed using Repeated Measures ANOVA followed by Bonferoni’s Post Hoc test. #Statistically significant difference (p<0.05) in reaction time when compared to pentazocine, *Statistically significant difference (p<0.05) in reaction time when compared to Distilled water n = 6. Penta = Pentazocine, CF= Chloroform fraction of the T. bracteolata, DW=Distilled water Carrageenan-Induced Paw Edema The fraction at doses of 200 and 400 mg/kg significantly (p< 0.05) decreased the paw edema size in rats at time 3, 4- and 5-hours post carrageenan induction when compared with the distilled treated group (Table III). 34 Sadam et al./Nig.J.Pharm. Res. 2020, S1:31-37 Table 3: Effect of Chloroform fraction of T.bracteolata on Carragenan-Induced Paw Edema Test in Rat Treat Dose Mean Paw Diameter in cm ment (mg/kg) (% Inhibition) 1h 2h 3h 4h 5h DW ml/kg 3.44±0.10 3.56±0.06 3.61±0.13 3.38±0.12 2.82±20.05 CF 100 CF 200 CF 400 Ibu 10 3.22±0.12 (6.39) 2.62±0.2 (19.14) 1.74±0. 10* (46.29) 1.88±0.21* (45.35) 3.04±0.17 (14.61) 2.60±0.19* (26.9) 1.75±0.11 (50.84) 1.86±0.06* (47.75) 2.61±0.13 (27.70) 2.55±0.14* (29.36) 1.73±0.03* (52.08) 1.74±0.08* (51.80) 2.50±0.13* (26.04) 2.51±0.1 (25.74) 1.61±0.04* (52.37) 1.54±0.07* (54.44) 2.40±0.06* (14.89) 2.32±0.08* (17.73) 1.34±0.03* (52.48) 1.42±0.04* (49.65) Values presented as Mean ± SEM. Data were analysed using Repeated Measures ANOVA followed by Bonferoni post hoc test. * p<0.05 significant difference compared to DW group, n = 5. Ibu = Ibuprofen, CF= Chloroform fraction of the T.bracteolata, DW= Distilled water. DISCUSSION Acetic acid-induced writhing test is a very sensitive model used for evaluating the peripheral antinociceptive activity of extract at a very low dose (Shumaia et al., 2014). The CF in graded doses administered significantly (p≤0.05) produced decrease in the number of writhes induced by acetic acid dose dependently with the highest percentage inhibition brought by the highest dose respectively. The intraperitoneal administration of acetic acid resulted in increased level of prostanoids (PGE 2 and PGF2α) and lipoxygenases (Derardt et al., 1980) in the peritoneal fluid thereby stimulating the nerve endings (pain) through; the release of pain mediators such as arachidonic acid via cyclooxygenase, the synthesis of chemo-sensitive niciceptors by endogenous substances such as bradykinins, prostaglandins (PGs), serotonin and histamine (Davies et al., 1984). The model is thought to involve in part local peritoneal receptors (Bentley et al., 1983). The CF extract may have interfered with these peritoneal receptors to bring about analgesia (Musa et al., 2009). The analgesic activities exhibited by the CF extract may occur through the process of inhibiting the production of those algogenic substances or its action on visceral receptors sensitive to acetic acid. The extract produced strong analgesic effect on the acetic acid-induced writhing in the same order of magnitude as that observed by piroxicam administration. Thermally induced hyperalgesia is a specific model for testing centrally mediated antinociception and the observed activity is selective for compounds acting through opioid receptor (Kaushik et al., 2012). The increase in the mean latency to pain by CF in the thermally-induced pain in mice suggests that the extract possesses centrally acting analgesic activity (Kaushiket al., 2012). At dose of 400 mg/kg, CF at 60 and 90 minutes exhibited greater activity when compared to the standard agent pentazocine at 10 mg/kg. This suggests that the extract may act via centrally mediated mechanism (Kaushik et al., 2012). The extract (100 mg/kg) at 30 minutes produced a significant effect (p<0.05) on the pain threshold on the test group when compared to the positive control, suggesting that the activity of the extract is dose dependent. The process of inflammation is multiphasic including; the increase in vascular permeability, leucocytes infiltration and the formation of granuloma. Carrageenan is a phlogistic agent that induces edema in the paw of experimental animals used as a model for testing anti-inflammatory activity (Chatterjee et al., 2015). The early phase (1 hour) of carrageenan induction in the sub-plantar region of the paw is mediated by the release of histamine, serotonin and kinins. The late phase (2-3 hours) is mediated by the release of prostaglandins and lysosome enzymes (Ahmed., 2011). CF extract contains flavonoids, several derivatives of flavonoids are known to exhibit anti-inflammatory and analgesic activities by inhibiting the enzyme prostaglandin synthetase, more specifically the endoperoxidase reducing the release of arachidonic acid (Derardt et al., 1980). Therefore, the anti-inflammatory activity exhibited by CF extract may be due to the inhibition of COX1 and COX2 that converts arachidonic acid to PGG2 and PGH2 leading to the production of thromboxane A2 (Burke et al., 2006). This process is similar to that of NSAIDs (Roberts and Morrow, 2001) thus validating the ethnomedical use of the plant in 35 Sadam et al./Nig.J.Pharm. Res. 2020, S1:31-37 achieving analgesia and treating inflammatory conditions. The result of the oral acute toxicity study of the CF extract shows that after the administration of 2000 mg/kg of the extract, no lethality and mortality were observed. The LD50 was therefore determined to be 2000 mg/kg which is relatively non-toxic. 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Received: October 2020 Accepted: December 2020 Telephone: +2347033185517 E-mails: sadam4rich@gmail.com 37