http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(9): 1208–1216 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.880487

ORIGINAL ARTICLE

Analgesic and anti-inflammatory effects of the methanol root extracts of some selected Nigerian medicinal plants Ismail O. Ishola1,2, Esther O. Agbaje1, Olufunmilayo O. Adeyemi1, and Rakesh Shukla2 Department of Pharmacology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria and 2Division of Pharmacology, Central Drug Research Institute, Lucknow, Uttar Pradesh, India

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1

Abstract

Keywords

Context: The roots of Alafia barteri Oliver (Apocynaceae), Combretum mucronatum Schumach (Combretaceae) and Capparis thonningii Schum (Capparaceae) are used in Traditional African Medicine to alleviate painful and inflammatory conditions. Objective: This study investigated the analgesic and anti-inflammatory effects of the methanol root extracts of Alafia barteri (MeAB), C. mucronatum (MeCM), and Capparis thonningii (MeCT). Materials and methods: Analgesic activity of the extracts (50, 100, and 200 mg/kg, p.o. 1 h) was evaluated using acetic acid-, formalin- and hot plate-induced pain while anti-inflammatory actions (100 or 200 mg/kg) were investigated using the carrageenan- and xylene-induced edema tests. Results: MeAB, MeCM, and MeCT (200 mg/kg) inhibited acetic acid-induced abdominal constriction by 55.07, 46.67, and 47.25%, respectively. In the formalin test, the index of pain inhibition of early and late phases was, respectively, 47.83 and 81.98% for MeAB, 56.10 and 63.81% for MeCM, and 42.84 and 63.29% for MeCT (200 mg/kg). MeAB and MeCT pretreatments significantly increased the reaction time by 46.67 and 25.53%, respectively, 120 min posttreatment in the hot-plate test. Naloxone (5 mg/kg, s.c.) pretreatment 15 min before extract administration, significantly (p50.05) reversed the analgesic effect of MeAB and MeCT in the formalin test. MeAB, MeCM, and MeCT showed significant anti-inflammatory activity with 60.44 and 30.39%, 63.74 and 58.08%, and 50.55 and 77.84% (200 mg/kg, 4 h), respectively, inhibition of paw and ear edema. Discussion and conclusion: The analgesic and anti-inflammatory effects of MeAB and MeCT involve an interaction with opioid pathway and/or inhibition of chemical mediators of pain and inflammation.

Alafia barteri, Capparis thonningii, Combretum mucronatum, formalin, naloxone, opioid pathway, phospholipase A2, xylene

Introduction Inflammation is a reaction of the body against an aggressive agent, characterized by vasodilatation, access of fluid and cells to the target tissue (Schmid-Scho¨nbein, 2006). One of the major signs of inflammation is the pain that can be triggered by direct stimulation of nociceptors or by the action of inflammatory mediators (Brenner & Krakauer, 2003). These mediators, for example, cytokines, histamine, serotonin, leukotrienes, and prostaglandins, increase the vascular permeability and migration of leukocytes to inflamed tissue (Brenner & Krakauer, 2003). The usual treatment of inflammatory pain is done by non-steroidal anti-inflammatory drugs, but not without adverse effects like gastrointestinal ulceration accompanied by anemia from the resultant blood loss, fluid retention, bronchospasm, and prolongation of

Correspondence: Rakesh Shukla, Ph.D., Division of Pharmacology, Central Drug Research Institute, Lucknow, Uttar Pradesh, India. Tel: +91 522 2612411 18x4420. Fax: +91 522 2623405. E-mail: [email protected]

History Received 23 April 2013 Revised 22 November 2013 Accepted 1 January 2014 Published online 28 March 2014

bleeding time (Derle et al., 2006). Plant extracts can be an important source of natural and safer drugs for the treatment of painful and inflammatory conditions. The current trend towards utilization of plant-derived natural remedies has created a dire need for accurate information on the uses, efficacy, safety, and quality of medicinal plant products. In this study, an attempt was made to evaluate the analgesic and anti-inflammatory activities of three medicinal plants used in the management of painful and inflammatory conditions by traditional healers in Southwest, Nigeria. Alafia barteri Oliver (Apocynaceae) is a high-climbing scandent shrub with small, pure white, or pink flowers (Irvine, 1961). It is used in ethnomedicine for the treatment of sickle cell anemia, rheumatism, eye infections, as febrifuge, as chew sticks, and for toothache (Burkill, 1985). The twining stem of A. barteri is used for the treatment of fever and inflammation (Burkill, 1985; Leeuwenberg, 1997; Nadkarni, 1976). In Coˆte d’Ivoire, the leaf infusion is used to treat malaria. The root decoction is taken to treat rheumatic pains in Nigeria (Burkill, 1985). Antifungal properties of the ethanol and water extracts of A. barteri leaves have been

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DOI: 10.3109/13880209.2014.880487

Analgesic and anti-inflammatory activities of Nigerian plants

reported (Adekunle & Okoli, 2002). A previous study by Hamid and Aiyelaagbe (2011) has revealed that the phytochemical constituents include saponins, reducing sugars, steroids, glycosides, flavonoids, and anthraquinones. However, no study has been carried out to isolate its phytoactive constituents. Combretum mucronatum Schumach (Combretaceae) is widely distributed in West Africa and could be found in the region of Savannah forest (Ogundare & Akinyemi, 2011). The plant is mainly found in Western Nigeria, especially during the rainy season. The plant is used in traditional African medicine for the treatment of various forms of illnesses. The leaves and roots are used in the traditional medicine for the treatment of wounds, cough, dysentery, arthritis, and as antihelmintic, antimicrobials, and antipyretic (Ogundare & Akinyemi, 2011; Sofowora, 1982). The roots, cut in small size, are boiled with Capsicum peppers (Solanaceae) or woodash and the concoction is drunk for chest pains, rheumatic pain, and gonorrhea (Ogundare & Akinyemi, 2011). An infusion of young leaves with natron is used as a vermifuge in Ghana (Irvine, 1961; Ogundare & Akinyemi, 2011). Phytochemical screening by Ogundare and Akinyemi (2011) showed that it contains saponins, cardiac glycosides, tannins, and anthraquinones, but its active constituent is yet to be isolated. Capparis thonningii Schum (Capparaceae) is a climbing or scrambling pricky shrub of the Savannah woodland indigenous to West Africa. In folk medicine, it is used to reduce swelling, cure cough, and blood spitting in the region of Ghana (Ainslie, 1937). It is also used to calm toothache and earache in southwest Nigeria. The root is used in Senegal to treat vaginal discharge and syphilis (Kerharo & Adam, 1974; Sama & Ajaiyeoba, 2006). The phytochemical analysis by Sama and Ajaiyeoba (2006) revealed the presence of alkaloids, saponins glycosides, cardiac glycosides, and steroidal nucleus. Moreover, further study will be carried out to isolate the phytoactive constituents responsible for the pharmacologic effects. Methanol was chosen as a solvent for the extraction based on our preliminary investigation which showed that the methanol extract was more effective than aqueous extract. To the best of our knowledge, there are no reports concerning the analgesic and anti-inflammatory activities of the methanol root extract of A. barteri, C. mucronatum, and C. thonningii. Hence, this study was carried out to provide scientific evidence to the folklore uses of these plants in the treatment of pain and inflammation using various animal models.

Materials and methods Plant materials The plant materials were collected based on traditional healers’ information. Fresh root of C. mucronatum and Capparis thonningii were collected in March 2009 from Obantoko, Abeokuta, Ogun State, Nigeria, while the root of A. barteri was collected in March 2009 from Ibadan, Oyo State, Nigeria. The plant materials were identified and authenticated by Mr. T.K. Odewo (formerly Senior Superintendent, Forestry Research Institute of Nigeria

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(FRIN), Ibadan, Oyo State, Nigeria), now in the Department of Botany and Microbiology, University of Lagos, Lagos, Nigeria, where voucher specimens were deposited for reference purposes. The herbarium voucher specimen numbers are LUH 2880, 2881, and 2889 for A. barteri, C. thonningii, and C. mucronatum, respectively. Extraction The roots were air-dried at room temperature. The air-dried roots (2 kg) were pulverized into coarse powder with a Waring commercial blender. The powdered roots were extracted five times, on each occasion with 9.6 l of methanol at room temperature for 48 h (with occasional shaking). The combined methanol extracts were filtered and the filtrates were concentrated to dryness under reduced pressure in BUCHI RotavaporÕ (New Castle, DE) at 40  C. They were further dried with a vacuum pump to remove moisture, giving 320, 114.28, and 248 g (16.00, 5.71, and 12.40% yield) of A. barteri, C. mucronatum, and C. thonningii, respectively. The extracts were dissolved in 0.5% v/v dimethylsulfoxide in normal saline and were administered to the animal by oral gavage. Experimental animals Sprague–Dawley male rats (140–170 g) and 8-week-old Swiss albino mice (20–30 g) of either sex were obtained from the Laboratory Animal Services Division of Central Drug Research Institute, Lucknow, India. The animals were kept in polyacrylic cages with five rats or six mice per cage and maintained under standard housing conditions (room temperature 28  C and humidity 60–65%) with a 12-h light and dark cycle. Food, in the form of dry pellets, and water were available ad libitum but food was not allowed from 12 h prior to and until completion of the experiments. Experiments were performed according to international ethical standards and the protocol was approved by the Research Ethics Committee of Central Drug Research Institute and CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals). The experimental procedures adopted in this study were in accordance with the United States National Institutes of Health Guidelines for Care and Use of Laboratory Animals in Biomedical Research (NIH, 2001). Drugs and chemicals Chemicals and drugs used in this work are: acetic acid, carrageenan, diazepam, quercetin, Folin-Ciocalteu’s phenol reagent, sodium carbonate, xylene, formalin (Sigma Aldrich, St. Louis, MO), methanol (Merck, Bangalore, India), diclofenac sodium (Novartis India Limited, Bangalore, India), and normal saline (UNIC Pharmaceuticals, Ogun, Nigeria). Acute oral toxicity test – fixed dose procedure The acute oral toxicity test was conducted using the fixed dose procedure according to OECD test guidelines on acute oral toxicity TG 420 as adopted on 17 December 2001. Fifteen female mice (25–30 g) were randomly selected from a group of 40 mice. Based on the recommendations of several expert meetings in 1999, testing in one sex (usually females)

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is generally considered sufficient for acute toxicity testing (OECD, 2001). The selected mice were divided into three groups of five mice each. Each group was kept separately in polyacrylic cages, feed but not water was withheld for 4 h before the test. A stepwise procedure using fixed doses of 300, 2000, and 4000 mg/kg/body weight per extract with 3 d in-between each dose. Each animal was observed for the first 10 min after treatment then every 30 min for the next 6 h for behavioral acute toxicity symptoms. The mice were further observed for up to 14 d following treatment for any signs of toxicity and mortality.

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Total phenolic content (TPC) The sample TPCs were measured according to the method of Singleton and Rossi (1965) with slight modifications. To determine the levels of TPC, 1 ml of each extract was combined with Folin–Ciocalteu’s phenol reagent and water 1:1:20 (v/v). The mixture was incubated for 8 min followed by the addition of 10 ml of 7% (w/v) sodium carbonate. After 2 h, the absorbance of each sample mixture was measured at 750 nm. The values of TPC were estimated by comparing the absorbance of each sample mixture with those of a standard response curve generated with quercetin. Results were expressed as mg quercetin per mg of extract. Pharmacological studies Antinociceptive test The test was performed as described by Koster et al. (1959). Mice (20–30 g, n ¼ 5 per group) were pretreated with 0.5% v/v dimethylsulfoxide in normal saline (10 ml/kg, p.o.), MeAB, MeCM, MeCT (50– 200 mg/kg, p.o.), and diclofenac (100 mg/kg, p.o.) 60 min before intraperitoneal injection of acetic acid (0.6% v/v, 0.1 ml/10 g body weight). The number of abdominal writhes (contraction of the abdominal muscle together with a stretching of the hind limbs) was cumulatively counted every 5 min for a period of 20 min. The antinociceptive activity was expressed as percentage inhibition of abdominal writhes (Ishola et al., 2011). Acetic acid-induced writhing test.

Formalin test. Mice fasted overnight were divided into eight

groups (n ¼ 5). The different groups of animals were treated with 0.5% v/v dimethylsulfoxide in normal saline (10 ml/kg, p.o.); MeAB, MeCM, and MeCT (100 or 200 mg/kg, p.o.), and morphine (10 mg/kg, s.c.). Sixty minutes after administration for the oral route or 30 min for the subcutaneous route, formalin (20 ml of 1% solution) was injected subplantar into the right hind paw of each mouse. The time (in seconds) spent in licking or biting the injected paw, indicative of pain, was recorded for each animal. The responses of the mice were observed for the first 5 min (neurogenic phase) and 15–30 min (inflammatory phase) post-formalin injection (Ishola et al., 2011). The percent pain inhibition was calculated using the following formula: Inhibition ð%Þ ¼

reaction time ½control  reaction time ½treated 100 reaction time ½control

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The hot-plate was used to measure response latencies according to the method described by Eddy and Leimbach (1953), with minor modifications. Mice of either sex were screened for response to thermally induced pain by placing them on (Columbus Analgesiometer, Columbus, OH) a hot plate maintained at 55  C. The time between the placement of the mouse on the hot plate and shaking or licking of the paws or jumping was recorded as the reaction latency. Mice with baseline latencies higher than 10 s were excluded from the study. Animals that reacted to the thermally induced pain in less than 10 s were divided into 13 groups (n ¼ 5); group 1: 0.5% v/v dimethylsulfoxide in normal saline (10 ml/kg, p.o.), group 2: morphine (10 mg/kg, s.c.), group 3: diclofenac (100 mg/kg, p.o.), group 4: nimesulide (100 mg/kg, p.o.), groups 5–7: A. barteri (50–200 mg/kg, p.o.), groups 8– 10: C. mucronatum (50–200 mg/kg, p.o.), and groups 11–13: C. thonningii (50–200 mg/kg, p.o.). The reaction latency was recorded at 30, 60, 90, 120, and 150 min post-drug treatment. The prolongation of the latency times comparing the values before and after the administration of the extract- or vehicletreated control was considered analgesic response (Ishola et al., 2012) Hot-plate test.

% MPE ðpercent maximum possible effectÞ ¼ ðtest  baselineÞ=ðcut  off  baselineÞ  100, where the test is the latency to respond after treatment; baseline is the latency to respond prior to treatment; and cutoff (10 s) is the preset time at which the test will be ended in the absence of a response. Possible involvement of the opioidergic pathway Animals were pretreated with a dose of antagonist that has been shown to inhibit the antinociceptive activity of drug in rodents 15 min before the oral administration of MeAB, MeCM, and MeCT (200 mg/kg, p.o.). The possible involvement of opioidergic pathway in the analgesic effect of the extracts was investigated through subcutaneous injection of naloxone (5 mg/kg) (Rajendran et al., 2000; Vidyalakshmi et al., 2010); 15 min post-treatment, the animals were given the extracts per os. One hour after the oral administration of the extracts, 1% formalin (20 ml, s.c.) was injected into the right hind paw. Each mouse was then returned to the observation cylinder and the nociceptive response was recorded for the first 5 min and at 15–30 min post formalin injection representing the early neurogenic and the late inflammatory phases, respectively. The percentage inhibition was calculated (Ishola et al., 2011). Anti-inflammatory test Carrageenan-induced rat paw edema This test was carried out using carrageenan as a phlogistic agent to induce paw edema in the right hind limb of rat, which serves as a model of acute inflammation (Winter et al., 1962). Sprague–Dawley male rats (140–170 g) were randomly divided into groups of six animals each, and were used after a 12-h fast but allowed free access to water except during the experiment. MeAB, MeCM, and MeCT (100 or 200 mg/kg/ body weight, p.o.), diclofenac 100 mg/kg, p.o. (reference

DOI: 10.3109/13880209.2014.880487

Analgesic and anti-inflammatory activities of Nigerian plants

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drug) and 0.5% v/v dimethylsulfoxide in normal saline (10 ml/kg, p.o.) were administered 1 h before subcutaneous injection of 100 ml of carrageenan (1% w/v in 0.9% normal saline) into the right hind paw. The needle is inserted to a depth of approximately 1 mm into the callus to deliver an accurate and uniform amount of carrageenan into the subplantar site. Paw volume was measured by means of a volume displacement method using a plethysmometer (Ugo-Basile, Varese, Italy) prior to the injection of carrageenan and thereafter at 1,2, 3, 4, 5, 6, and 24 h. Edema was expressed as the change in paw volume (ml) after carrageenan injection relative to the pre-injection value for each animal. Percentage inhibitions of edema were calculated (Adeyemi et al., 2008; Ishola et al., 2012). Xylene-induced ear edema Mice were randomly allotted to groups of five animals each. Thirty minutes after the oral treatment of mice with normal saline (10 ml/kg), prednisolone (10 mg/kg), MeAB, MeCM, and MeCT (100 or 200 mg/kg/body weight, p.o.), edema was induced in each mouse by applying 30 ml of xylene to the inner surface of the right ear. Fifteen minutes post xylene application, the animals were euthanized under ether anesthesia and both ears were cut off, sized, and weighed. The mean of the difference between the right and left ears was determined for each group and the percentage inhibition was calculated (Akindele & Adeyemi, 2007; Nunez Guillen et al., 1997). Spontaneous locomotor activity Each animal was observed for 5 min after a period of 2 min for acclimatization in Optovarimex activity meter (Columbus Inc., Columbus, OH). MeAB, MeCM, and MeCT (200 mg/kg, p.o.) were administered 30 min after the initial observation (pretreatment counting) and 60 min after drug treatment. The animal was placed in the activity meter for counting the locomotor activity. Neuromuscular coordination – rotarod test The effect of the extracts on coordinated motor movements was assessed using the rotarod test (Columbus Rotamex-5, Columbus, OH). Mice (20–30 g) were trained for 3 d (three trials per day) before the test. The animals were trained on the rota-rod at a fixed speed of 20 rpm until they could remain on the apparatus for 5 min (only the animals that could stay on the rotating rod for 5 min were divided into groups of six mice each). On the day of the experiment, mice were placed on the rotarod apparatus (20 rpm for 5 min) and the latency of falling was measured before and at 30 and 60 min post-treatment with diazepam (5 mg/kg, p.o.), MeAB, MeCM, and MeCT (200 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.). Results were expressed as an average time for mice to stay on the rotarod in each group. Statistical analysis Results obtained were expressed as mean ± SEM. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc multiple comparison tests, or a two-way ANOVA

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followed by Bonferroni post hoc multiple comparison tests. The statistical analysis was performed with the aid of Graphpad prism version 5 (GraphPad Software, San Diego, CA).

Results Acute toxicity test Oral administration of MeAB, MeCM, and MeCT up to 4 g/kg neither induced mortality nor toxic behaviors. Total phenolic content MeAB, MeCM, and MeCT were found to contain 0.0019, 0.0155, and 0.0076 polyphenols (mg)/mg extract, respectively. Acetic acid-induced mouse writhing test Oral administration of MeAB (100 or 200 mg/kg) significantly (p50.01; p50.001) reduced the mean number of writhes from 68.80 ± 5.72 in control to 36.80 ± 2.63, and 31.40 ± 3.71 (46.38 and 55.07% inhibition, respectively). Similarly, the pretreatment of mice with MeCM (100 or 200 mg/kg) significantly (p50.01) reduced the mean number of abdominal constrictions from 68.70 ± 5.72 in vehicletreated control to 36.80 ± 2.63 (46.67% inhibition) at 200 mg/ kg. In this study, oral administration of MeCT (200 mg/kg) significantly (p50.01) reduced the mean number of writhes to 36.40 ± 3.72 (47.25% inhibition). All the three extracts elicited a non-dose-related reduction in the occurrence of abdominal constrictions. However, the antinociceptive effect of the reference standard diclofenac (86.67%) was significantly (p50.01) higher than that of the extracts (Figure 1). Formalin test Oral administration of MeAB, MeCM, and MeCT elicited significant (p50.05) reduction in the duration of paw licking and biting from 59.80 ± 5.49 s in vehicle-treated control to 31.20 ± 5.05, 26.25 ± 4.77, or 34.18 ± 2.82 s, respectively, at 200 mg/kg, but significantly (p50.05) lower than the analgesic effect produced by morphine (5.02 ± 0.60 s) in the early phase (Table 1). Similarly, in the inflammatory phase, acute oral pretreatment with MeAB, MeCM, and MeCT significantly (p50.001) reduced the duration of nociceptive reaction from 116.60 ± 11.84 s in control treated to 21.00 ± 3.67, 42.20 ± 5.23, and 42.80 ± 5.78 s, respectively, at 200 mg/kg, which was comparable, but significantly (p50.05) lower than the effect of opioid analgesic (morphine) that reduced the duration of paw biting to 2.32 ± 0.86 s (98.94% inhibition) (Table 1). Possible involvement of opioidergic pathway Subcutaneous injection of naloxone (5 mg/kg) significantly (p50.05) reversed MeAB-induced analgesia in both early and late phases of formalin test (Table 1). However, naloxone pretreatment failed to inhibit MeCT-induced analgesia in the inflammatory phase of formalin test; interestingly, naloxone pretreatment could not prevent MeCM-induced

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analgesia in both the early and late phases of formalin test in mice (Table 1).

In the hot-plate test, neither diclofenac (100 mg/kg, p.o., non-selective cyclooxygenase inhibitor) nor nimesulide (100 mg/kg, p.o., cyclooxygenase II inhibitor) induced significant antinociceptive effects on reaction time during 150 min of observation. However, subcutaneous pretreatment with morphine induced significant analgesia, as shown by the delays in reaction time with a peak effect (6.20 ± 0.73 s, 44.1% MPE) of 90 min post treatment (Table 2). The two-way ANOVA showed that oral administration of MeAB, MeCM, and MeCT (50, 100, and 200 mg/kg, p.o.) elicited significant analgesia in the hot-plate test as evidenced by an increase in the reaction latency (Table 2) in comparison with vehicle treated control. Pretreatment with MeAB (50, 100, and 200 mg/kg) produced significant (p50.05) time-dependent increase in mean reaction latency with peak effects 35.29, 46.67, and 100 80 60

b

40 20

c c c

c

c c

α c

0 Ve Di hic cl le of M ena e c M AB eA 50 M B1 eA 0 0 M B2 eC 00 M M eC 5 0 M M e C 10 M 0 M 20 e 0 M CT eC 50 M T1 eC 0 T 0 20 0

mean number of writhes in 20 mins

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Hot-plate test

Figure 1. Effect of methanol root extracts of A. barteri, C. mucronatum, and C. thonningii against acetic acid-induced mouse writhing test. Values are expressed as mean ± SEM (n ¼ 5). The level of statistical significance was measured using one way ANOVA followed by Tukey post hoc multiple comparison test. bp50.01, cp50.001 versus vehicletreated control; ap50.05 versus A. barteri, C. mucronatum, and C. thonningii 200 mg/kg.

60.29% maximum possible effects at 90, 120, and 150 min post-treatment, respectively. However, MeCM (50–200 mg/kg, p.o.) treatment failed to increase (p40.05) the reaction latency with a maximum possible effect of 27.54%, 30 min post treatment at 50 mg/kg (Table 2). In this study, acute oral administration of MeCT produced significant (p50.05) time-dependent increase in mean reaction latency with peak significant maximum possible effect 28.99%, 60 min post treatment at 100 mg/kg. The analgesic effect produced by MeAB was comparable with that of morphine with 42.02% maximum possible effect, 60 min post treatment (Table 2). Carrageenan-induced rat paw edema Antiedematogenic activities of MeAB, MeCM, and MeCT are presented in Table 3. Injection of 100 ml of carrageenan into right hind paw of rats produced time-dependent increase in paw size which peaked at 5 h (1.13 ± 0.14 ml mean change in paw volume) post-phlogistic agent injection. Pretreatment with diclofenac (standard reference drug) produced timedependent significant inhibition of edema formation with a peak effect of 75.29% inhibition 24 h post-phlogistic agent injection. Similarly, oral administration of MeAB, MeCM, and MeCT (100–200 mg/kg) produced dose-related and timedependent significant (p50.05, p50.01, p50.001) suppression of inflammation in the middle and late phases when compared with the vehicle-treated control group with peak effects (82.35, 63.64, and 56.57 % inhibition) from the 3rd to 24th h, respectively, post-phlogistic agent administration at 200 mg/kg. These effects were less than but not significantly different (p40.05) from that produced by diclofenac (66.67% inhibition) (Table 3). Xylene-induced ear edema Oral administration of MeAB non-significantly reduced ear edema development (p40.05) with the peak effect (30.33% inhibition) at 200 mg/kg. However, MeCT and MeCM (200 mg/kg) significantly (p50.05, p50.001, respectively) and dose-dependently reduced ear edema formation from 33.40 ± 3.75 mg in the control group to 14.00 ± 4.36 or 7.40 ± 2.11 mg (Table 4). The degree of ear edema inhibition produced by prednisolone (10 mg/kg; p.o.) treatment was

Table 1. Effect of selected medicinal plants against formalin-induced pain in mice. 0–5 min

15–30 min

Treatment

Dose (mg/kg)

Duration of paw licking

% inhibition

Duration of paw licking

% inhibition

Vehicle Morphine A. barteri

10 10 100 200 100 200 100 200 10 200 200 200

59.8 ± 5.5 5.0 ± 0.6 48.6 ± 9.0 31.2 ± 5.1 56.3 ± 5.0 26.3 ± 4.8 51.4 ± 8.0 34.2 ± 2.8 54.0 ± 6.9 43.6 ± 3.8 30.4 ± 5.6 47.2 ± 3.7

– 90.5c,g 18.7 47.8a 5.9 56.1b 14.1 42.8a 9.7** 27.1 49.2a 21.1

116.6 ± 11.8 2.3 ± 0.9 69.4 ± 8.2 21.0 ± 3.7 99.8 ± 10.4 42.2 ± 5.2 100.0 ± 6.4 42.8 ± 5.8 85.6 ± 9.0 86.6 ± 7.3 34.6 ± 5.0 69.4 ± 2.5

– 98.0c 40.5a 82.0c 14.4 63.8c 14.2 63.3c 26.6 25.7## 70.3c 40.5c

C. mucronatum C. thonningii Naloxone Naloxone Naloxone Naloxone

+ + + +

morphine A. barteri C. mucronatum C. thonningii

Values are expressed as mean ± SEM (n ¼ 5), ap50.05, bp50.01, cp50.001 versus vehicle-treated control group; **p50.01 versus morphine 10 mg/kg treated group; gp50.05 versus A. barteri, C. mucronatum, and C. thonningii (200 mg/kg) treated group. ##p50.01 versus A. barteri 200 mg/kg treated group (a one-way ANOVA followed by Tukey’s post hoc multiple comparison tests).

Analgesic and anti-inflammatory activities of Nigerian plants

DOI: 10.3109/13880209.2014.880487

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Table 2. Time course of the effect of selected medicinal plants against hot plate-induced pain. Reaction latency (s) Treatment

Dose (mg/kg)

0 min

30 min

Vehicle Morphine Diclofenac Nimesulide A. barteri

10 10 100 100 50 100 200 50 100 200 50 100 200

2.5 ± 0.2 2.0 ± 0.2 2.5 ± 0.1 2.5 ± 0.3 2.6 ± 0.2 2.2 ± 0.2 1.9 ± 0.2 2.1 ± 0.2 1.7 ±0.1 1.6 ± 0.1 2.5 ± 0.2 2.1 ± 0.3 1.9 ± 0.2

3.2 ± 0.5 4.6 ± 0.3 (20.6) 2.1 ± 0.2 (0.0) 2.4 ± 0.5 (0.0) 4.7 ± 1.0 (22.1) 3.3 ± 0.8 (1.5) 2.7 ± 0.5 (0.0) 2.4 ± 0.2 (0.0) 2.5 ± 0.3 (0.0) 2.6 ± 0.4 (0.0) 4.0 ± 0.9 (27.5) 3.13 ± 0.5 (0.0) 3.6 ± 0.7 (5.9)

C. thonningii

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C. mucronatum

60 min 3.1 ± 0.4 6.0 ± 0.7 3.2 ± 0.3 3.1 ± 0.6 5.5 ± 1.0 5.8 ± 0.7 5.2 ± 1.2 3.1 ± 0.2 5.1 ± 0.2 4.5 ± 1.0 4.3 ± 0.5 4.0 ± 1.3 3.9 ± 0.8

90 min 3.2 ± 0.4 6.2 ± 0.7 2.7 ± 0.4 2.7 ± 0.3 5.6 ± 0.9 4.8 ± 0.8 7.3 ±1.3 4.0 ± 0.5 4.8 ± 0.4 3.4 ± 0.5 3.6 ± 0.3 3.0 ± 0.7 3.5 ± 0.2

(42.0b) (0.0) (0.0) (34.8) (39.1a) (30.4a) (0.0) (29.0a) (20.3) (15.9) (11.6) (10.3)

120 min

(44.1b,g) (0.0) (0.0) (35.3) (23.5) (60.3c) (11.8) (23.5) (2.9) (5.9) (0.0) (4.4)

2.5 ± 0.2 4.5 ± 1.0 3.3 ± 0.4 3.2 ± 0.6 3.7 ± 0.5 6.0 ± 0.7 4.4 ± 0.2 4.1 ± 0.5 4.3 ± 0.6 3.5 ± 0.5 3.7 ± 0.9 2.6 ± 0.3 3.4 ± 0.7

(26.7a) (10.7) (9.3) (16.0) (46.7b) (25.3a) (21.3) (25.3a) (13.3) (16.0) (1.5) (12.0)

150 min 2.2 ± 0.2 2.5 ± 0.4 2.4 ± 0.4 2.0 ± 0.3 3.7 ± 0.3 3.2 ± 0.6 2.6 ± 0.6 2.6 ± 0.4 2.3 ± 0.5 2.6 ± 0.7 3.9 ± 0.6 2.3 ± 0.4 3.6 ± 0.6

(3.9) (2.6) (0.0) (19.2) (12.8) (5.1) (5.1) (1.5) (5.1) (21.8) (1.5) (18.0)

Values are expressed as mean ± SEM (n ¼ 6). a,b,cSignificant increase in reaction latency: ap50.05, bp50.01, cp50.001 versus vehicle-treated control group,  p50.05 versus C. mucronatum 200 mg/kg (a two-way ANOVA followed by Bonferroni post hoc multiple comparison test). Values in parenthesis represent percentage maximum possible effect (%MPE).

Table 3. Effect of selected methanol root extracts against carrageenan-induced paw oedema in rats. Change in paw volume (ml) Treatment Vehicle Diclofenac A. barteri C. mucronatum C. thonningii

Dose (mg/kg) 10 100 100 200 100 200 100 200

1h 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 0.3 ± 0.0 0.3 ± 0.0 0.5 ± 0.1 0.6 ± 0.1 0.4 ± 0.1

(32.5) (25.0) (29.2) (4.2) (31.3) (17.9) (20.8)

2h 0.7 ±0.1 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 0.6 ± 0.1

3h (25.9) (24.6) (33.9) (10.8) (16.9) (5.9) (10.8)

0.9 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.7 ± 0.1

4h

(34.4) (47.2a) (49.4a) (49.4b) (49.4b) (43.6a) (43.8a)

0.9 ±0.1 0.5 ± 0.1 0.4 ± 0.1 0.4 ± 0.0 0.7 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.9 ± 0.1

(40.7a) (58.2c) (60.4c) (60.4c) (63.7c) (52.3b) (50.6b)

5h 0.9 ±0.1 0.5 ± 0.1 0.3 ± 0.2 0.3 ± 0.0 0.7 ± 0.2 0.4 ± 0.1 0.5 ± 0.1 0.9 ± 0.1

6h

(40.7a) (66.7c) (67.8c) (60.0c) (65.6c) (50.0b) (56.7b)

0.8 ± 0.1 0.5 ± 0.1 0.3 ± 0.2 0.3 ± 0.0 0.6 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 0.8 ± 0.1

(43.7a) (65.9c) (65.9c) (58.5b) (63.4b) (58.1b) (59.8b)

0.6 ± 0.1 0.2 ± 0.0 0.2 ± 0.2 0.1 ± 0.0 0.3 ± 0.0 0.1 ± 0.0 0.2 ± 0.1 0.4 ± 0.1

(63.5a) (73.7b) (77.2a) (75.4a) (50.9) (74.4a) (79.0b)

Values are expressed as mean ± SEM. a,b,cSignificant decrease in paw volume: ap50.05; bp50.01; cp50.001 versus vehicle-treated control group (a two-way ANOVA followed by Bonferonni post hoc multiple comparison test. Values in parenthesis represent the percentage inhibition of edema development following intraplantar injection of carrageenan.

comparatively similar to the anti-edema effect of MeCT and MeCM (200 mg/kg) (Table 4).

Table 4. Effect of selected medicinal plants against xylene-induced ear edema in mice.

Spontaneous locomotor activity The spontaneous locomotor activity was not significantly (p40.05) affected following the oral administration of MeAB and MeCM (200 mg/kg). However, oral administration of MeCT (200 mg/kg) significantly (p50.05) reduced total and ambulatory movement but not significant (p40.05) in vertical movement (Table 5).

Treatment Vehicle Prednisolone A. barteri C. thonningii C. mucronatum

Rotarod test MeAB, MeCM, and MeCT (200 mg/kg, p.o.) did not alter the motor performance of animals subjected to the rotarod task in comparison with the control group (vehicle, p.o.). Average values in the rotarod test for control, MeAB, MeCM, and MeCT (200 mg/kg, p.o.) and diazepam (5 mg/kg) were 300 s ± 0.00, 300 s ± 0.00, 273 s ± 22.34, 300 s ± 0.00, and 41.80 s ± 4.97, respectively (Figure 2).

Discussion This study was aimed at evaluating the scientific basis for the traditional use of A. barteri, C. mucronatum, and

Dose (mg/kg)

increase in ear weight (mg)

Inhibition (%)

10 10 100 200 100 200 100 200

33.4 ± 3.8 21.6 ± 1.8 30.6 ± 6.1 23.3 ± 2.1 15.5 ± 0.9 14.0 ± 4.4 23.8 ± 8.3 7.4 ± 2.1

35.3* 8.4 30.4 53.6* 58.1* 28.7 77.8***

Values are expressed as mean ± SEM. *Significant decrease in ear edema: *p50.05, ***p50.001 versus vehicle-treated control group (a one-way ANOVA followed by Tukey’s post hoc multiple comparison test).

C. thonningii against various painful and inflammatory conditions. Findings from this study showed that acute oral administration of MeAB, MeCM, and MeCT demonstrated analgesic and anti-inflammatory effects. In addition, the median lethal doses (LD50) of the extracts were estimated to be more than 4 g/kg. According to the OECD test guidelines

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Table 5. Effect of methanol root extracts on spontaneous locomotor activity in mice. Before Treatment Vehicle A. barteri C. mucronatum C. thonningii

After

Dose (mg/kg)

Total

Ambulatory

Vertical

Total

Ambulatory

Vertical

10 200 200 200

534.4 ± 22.7 616.5 ± 123.6 529.8 ± 61.6 529.0 ± 85.5

398.4 ± 34.6 463.5 ± 97.1 385.0 ± 54.8 383.8 ± 60.4

23.4 ± 4.5 25.8 ± 3.7 23.2 ± 5.7 16.2 ± 2.2

452.6 ± 19.1 340.5 ± 67.8 325.2 ± 94.0 208.8 ± 13.9

344.6 ± 37.8 242.3 ± 48.7 232.8 ± 71.9 133.2 ± 17.2

19.8 ± 2.5 19.8 ± 4.0 15.4 ± 2.2 13.2 ± 2.4

Figure 2. Effect of methanol root extracts of A. barteri, C. mucronatum, and C. thonningii on muscle coordination in rotarod test in mice. Values are expressed as mean ± SEM (n ¼ 6). ***p50.001 versus vehicle-treated control group. Statistical level of significance was analyzed using a one way ANOVA followed by Tukey post hoc multiple comparison test.

Mean duration of muscle coordination (s)

Vehicle 10 ml/kg MeCM 200 mg/kg MeCT 200 mg/kg MeAB 200 mg/kg Diazepam 5 ml/kg

400 300 200 100 ***

***

(2001) on acute oral toxicity TG 420, no dose-related toxicity should be considered above 4 g/kg body weight. In view of this assertion, the methanol root extracts of MeAB, MeCM, and MeCT can be considered safe when administered via the oral route. To estimate the antinociceptive property of plants used in traditional medicine, it is essential to employ different tests which differ in stimulus quality, intensity, and duration (Tjolsen & Hole, 1997). In the present study, the analgesic property of the selected Nigerian medicinal plants was investigated using the acetic acid mouse writhing, formalin, and hot-plate models of pain. The acetic acid mouse writhing and formalin-induced paw licking tests are often used to distinguish between central and peripheral analgesic actions (Nunez Guillen et al., 1997; Umukoro & Ashorobi, 2007) while the hot-plate test is used to detect centrally acting analgesics (Nunez Guillen et al., 1997). The extracts significantly inhibited the abdominal constriction induced by acetic acid in mice. Acetic acid has been shown to cause an increase in the level of PGE2 and PGF2a in peritoneal fluids (Deraedt et al., 1980; Rahman et al., 2005). In addition, previous studies have shown that acetic acid stimulates the vanilloid receptor (VR1) and bradykinin B2 receptor in the pathway comprising sensory afferent C-fibers (Ikeda et al., 2001). This study showed that the methanol root extract of A. barteri, C. mucronatum, and C. thonningii produced significant analgesic effect and this effect may stem from their ability to interfere with the synthesis or release of those endogenous substances (arachidonic acid metabolites; that sensitize and activate peripheral nociceptors) or desensitization of the nerve fibers involved in the pain transmission pathway (Jia et al., 2008). Our results suggest a peripherally mediated analgesic

m 60

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0

30

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Values are expressed as mean ± SEM (p40.05). Statistical level of significance by a two-way ANOVA followed by Bonferonni post hoc test.

activity based on the association of the model with the stimulation of peripheral receptors (Zakaria et al., 2008). It is important to note that other types of analgesics, such as the opioids, are also effective in this test without inhibiting prostaglandins synthesis. Hence, formalin-induced nociception was carried out to distinguish between the central and the peripheral antinociceptive action. The formalin test measures the ability of a drug to reduce the duration of persistent pain generated by injured tissue (Tjolsen et al., 1992). The first phase results from the direct stimulation of nociceptors, whereas the second phase involves a period of sensitization during which inflammatory phenomena occur (Le Bars et al., 2001). In this study, the methanol root extracts of A. barteri, C. mucronatum, and C. thonningii decreased the licking time in both phases, but the effect was more significant in the second phase. A decrease in licking time in both phases is characteristic of drugs that act centrally and indicates a possible interaction with opioid receptors. Opioid analgesics possess antinociceptive effect in both phases, although the first phase is more sensitive to these substances. In contrast, NSAIDs, such as ibuprofen, aspirin, seem to suppress only the second phase (Hunskaar & Hole, 1987). This observation suggest a more preferential and predominant effect of A. barteri, C. mucronatum, and C. thonningii on inflammatory pain. The predominant effect of the extracts in the inflammatory phase of formalin test indicates that their anti-inflammatory activity is an important one, and corroborates the results observed in the carrageenan-induced paw edema model where the extracts showed a potent anti-inflammatory activity. The pretreatment of mice with naloxone (non-selective opioid antagonist) significantly reversed the antinociceptive effect of

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DOI: 10.3109/13880209.2014.880487

Analgesic and anti-inflammatory activities of Nigerian plants

morphine and A. barteri but slightly reversed the antinociceptive effect of C. thonningii. However, naloxone failed to reverse the antinociceptive effect of C. mucronatum in the formalin test ruling out the involvement of opioidergic pathway in its analgesic effect. These suggest that A. barteri and C. thonningii may act through opioidergic pathway but a different mechanism of action for C. mucronatum is required for further investigation. The extent of the extracts’ central analgesic effects was confirmed in the hot-plate test. The hot-plate test produces two kinds of behavioral response, paw licking and jumping. Both are considered to be supraspinally integrated responses (Chapman et al., 1985; Chavan et al., 2012). Oral administration of diclofenac (non-selective cyclooxygenase inhibitor) and nimesulide (cyclooxygenase II inhibitor) failed to increase pain threshold in the hot-plate test. However, acute oral administration of A. barteri and C. thonningii increased pain threshold in the hot-plate test which was similar to the effect of morphine, a centrally acting analgesic drug but C. mucronatum failed to produce significant increase in reaction latency in the hot-plate test which was consistent to what was observed in the formalin test. This confirmed the central and peripheral analgesic mechanisms of A. barteri and C. thonningii possibly mediated through the opioidergic system (Chavan et al., 2012; Millan, 1994). The extracts showed significant inhibition of inflammation in both the carrageenan-induced rat paw swelling and xyleneinduced ear edema models. The results obtained from carrageenan-induced paw edema which is a valid animal model for assessing acute anti-inflammatory activity also confirmed the anti-edematogenic effect of A. barteri, C. mucronatum, and C. thonningii. Carrageenan-induced inflammation consists of three phases: early phase involves the production of histamine, serotonin, nitric oxide, and bradykinin, i.e., 1 h post phlogistic injection; second phase (at 2 h) mediated by kinins, leukotrienes, platelet-activating factor, and possibly cyclooxygenase products; and a third phase (3–24 h; late phase) primarily from the formation of pro-inflammatory prostanoids and nitric oxide (synthesized by the inducible nitric oxide synthase isoform), cytokines, neutrophil infiltration, and the production of neutrophilderived free radicals, such as hydrogen peroxide, superoxide, and OH radicals (Bilici et al., 2002; Di Rosa et al., 1971; Handy & Moore, 1998; Salvemini et al., 1996). In this study, A. barteri, C. mucronatum, and C. thonningii showed significant attenuation of edema development in the middle phase and more pronouncedly in the late phase of carrageenan-induced inflammation. This suggests that the extracts act by inhibiting the release and/or actions of kinins, pro-inflammatory prostanoids, and inducible nitric oxide synthase isoform (Bilici et al., 2002; Salvemini et al., 1996). The ‘‘late’’ phase inhibitory effect of these extracts depends on the inhibition of hind paw prostanoids formation presumably by an effect on COX-2 activity, which is likely to be the predominant isoform at this stage in the response (Seibert et al., 1994; Vane, 1998). The possibility that A. barteri, C. mucronatum, and C. thonningii may, additionally, reduce hind paw constitutive COX-1 activity to bring about an anti-edema effect in the ‘‘early’’

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phase is also suggested. However, further study will be carried out to determine cyclooxygenase inhibitory activity of the extracts. The xylene-induced ear edema model is useful for the evaluation of anti-inflammatory steroids, especially those inhibiting phospholipase A2 (Adeyemi et al., 2008; Zaninir et al., 1992). The effectiveness of the extracts in these two models suggests that they produced their anti-inflammatory effect by either inhibiting the synthesis, release, or action of inflammatory autacoids (Adeyemi et al., 2008). The antiinflammatory effect of the extracts was comparatively higher than those produced by diclofenac and prednisolone in carrageenan- and xylene-induced edema, respectively. At therapeutic doses, the extracts neither affected locomotor activity nor neuromuscular coordination. The preliminary phytochemical screening revealed the presence of flavonoids, saponins, glycosides, and anthraquinones in the extracts of A. barteri, C. mucronatum, and C. thonningii (Hamid & Aiyelaagbe, 2011; Ogundare & Akinyemi, 2011; Sama & Ajaiyeoba, 1998). The presence of one or combination of phytochemicals such as flavonoids, saponins, glycosides, and tannins in these extracts (Larkins & Wynn, 2004; Raaman, 2006; Ma et al., 2006) have been reported to possess analgesic and/or anti-inflammatory activities. Thus, the presence of these phytochemicals in the extracts may suggest that these active principles may be responsible for the potential antinociceptive and anti-inflammatory potentials of the tested extracts.

Conclusion The results of the present study suggest that A. barteri, C. mucronatum, and C. thonningii possess analgesic and antiinflammatory effects through an interaction with opioidergic pathway and/or inhibition of inflammatory autacoids, respectively, supporting the use of these plant species in folk medicine in the treatment of painful and inflammatory conditions.

Acknowledgements The provision of research facilities by Central Drug Research Institute (CDRI), Lucknow, Uttar Pradesh, India, is duly acknowledged.

Declaration of interest No conflict of interest to disclose. Authors sincerely acknowledge Third World Academy of Science (TWAS) and Council for Scientific and Industrial Research (CSIR) for the postgraduate Fellowship given to Ishola Ismail O.

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