PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1855–1860 (2014) Published online 11 August 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5212 Antiinflammatory Properties of the Stem-bark of Anopyxis klaineana and its Major Constituent, Methyl Angolensate Evelyn A. Mireku,1 Abraham Y. Mensah,1 Merlin L. K. Mensah,1 Derek A. Tocher2 and Solomon Habtemariam3* 1Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 2Department of Chemistry, University College London, 20 Gordon Street, London, UK 3Pharmacognosy Research Laboratories Medway School of Science, University of Greenwich, Central Avenue, Chatham-Maritime, Kent ME4 4TB, UK Anopyxis klaineana (Pierre) Engl. (Rhizophoraceae) is one of the reputed West African folkloric medicines that has never been investigated for its pharmacological effects or phytochemical constituents. In the present study, the antiinflammatory properties of the stem-bark extracts were evaluated using the carrageenan-induced paw oedema model in chicks. The petroleum ether, ethyl acetate and methanol extracts all showed a time and dose-dependent antiinflammatory effect over the 5-h observation period. Phytochemical analysis of the most active extract (methanol extract) yielded the principal constituent that was identified as methyl angolensate through extensive spectroscopic and X-ray analysis studies. Although slightly less potent (ED50, 4.05 ± 0.0034 mg/kg, orally) than the positive control, diclofenac (ED50, 2.49 ± 0.023, intraperitoneally n = 5), this first ever compound isolated from A. klaineana showed promising antiinflammatory activity that may account to some of the reported medicinal uses of the plant. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Anopyxis klaineana; Rhizophoraceae; methyl angolensate; antiinflammatory; carrageenan. such as gonorrhoea and as an enema to relieve stomach INTRODUCTION aches. The powdered bark is also applied topically for treating bronchitis and pneumonia, skin infections and Inflammation is a necessary host defence mechanism to deep wounds. In Ghana, the leaf decoction is also used tissue injury, infection or other cellular/biochemical dam- to treat malaria (Burkill, 1997; Oteng-Amoako and ages. In an acute inflammatory response, the immune reac- Essien, 2011; Asase et al., 2012). To the best of the authors’ tions to tissue damage are generally short-lived and knowledge, neither the phytochemical content nor the bio- ultimately lead to the restoration of normal tissue architec- logical activities of A. klaineana have ever been studied. In ture and function. In an unregulated inflammatory response the present communication, the antiinflammatory activities that is often characterized by abnormally high level of leu- of stem-bark of A. klaineana and its principal constituent, cocyte trafficking, however, extensive tissue damage leads methyl angolensate, are reported. to pathologies such as asthma, arthritis, atherosclerosis, inflammatory bowel disease, ischaemia-reperfusion injury, multiple sclerosis and sepsis (Habtemariam, 2010). Owing to the high demand of novel drugs for combating such MATERIALS AND METHODS chronic disease conditions, researches from many laborato- ries including ours have been focussing on medicinal plants that are reported to display antiinflammatory properties Chemicals. All organic solvents used for the experiments (Habtemariam, 2001, 2010; Woode et al., 2008; Mensah were of analytical grade and obtained from BDH et al., 2011). Laboratory Supplies (Merck Ltd, Lutterworth, UK). Anopyxis klaineana (Pierre) Engl. (Rhizophoraceae) is The standard reference drug, diclofenac, was purchased an evergreen, medium-sized to large-sized West African from Troge (Hamburg, Germany), whereas all other tree known locally as kokoti in Ghana (Sprague and chemicals were obtained from Sigma-Aldrich Company Boodle, 1909), bobenkusu in DR Congo, Noudougou in Ltd (Poole, Dorset, UK). Cameroon and bobioa in Cote d’Ivoire (Burkill, 1997). The stem-bark decoction is traditionally used to treat joint aches, kidney pains, sexually transmitted infections Collection and processing of the plant material. The stem-bark of A. klainenea was collected in November 2013 from a forest on the hills of Kwahu-Asakraka * Correspondence to: Solomon Habtemariam, Pharmacognosy Research ′ ′ ′ ′ Laboratories, Medway School of Science, University of Greenwich, Central (lat (DMS) 6°37 42.96 N, long (DMS) 0°41 21.00 W, Avenue, Chatham-Maritime, Kent ME4 4TB, UK. altitude 538 m), which was located about 30 km from E-mail: [email protected] Nkawkaw in the Eastern region of Ghana. The plant Received 21 May 2014 Revised 15 July 2014 Copyright © 2014 John Wiley & Sons, Ltd. Accepted 18 July 2014 1856 E. A. MIREKU ET AL. was authenticated at the Department of Herbal Medi- inhibition of oedema was calculated as [(AUC control cine, KNUST, and voucher specimen (No. KNUST/ AUC treatment)/AUC control] × 100. Differences in AK1/2013/S005) deposited at the department’s herbar- AUCs were analysed by one way analysis of variance ium. The plant material was gently washed with water, followed by Student Newman–Kuels’ post test; p < 0.05 dried at room temperature and powdered using a me- was considered statistically significant. Doses and con- chanical grinder. centrations responsible for 50% of the maximal effect (EC50) for each drug/extract were determined with the following nonlinear regression (three-parameter logistic) À À Extraction of the plant material. The powdered plant equation: Y = a (a + b)/(1 + 10 (log EC50 X)), where material (500 g) was Soxhlet-extracted successively with X is the logarithm of dose and Y is the response. Y starts 1500 mL each of petroleum ether, ethyl acetate and at a (the bottom) and goes to b (the top) with a sigmoid methanol. After removal of the solvents under reduced shape. Graph Pad Prism for Windows version 5.0 (Graph pressure, the petroleum ether, ethyl acetate and metha- Pad Software, San Diego, USA) was used for all statistical nol extracts were obtained with the yield of 0.88%, analyses, and all data are presented as mean and standard 2.91% and 6.09% (w/w), respectively. error of mean values. Animals. One-day-old chicks were obtained from Akate Isolation of the principal constituent of methanol extract Farms, Kumasi, Ghana, and were housed in stainless of the stem-bark. The crude extract (20 g) was loaded steel cages (34 × 57 × 40 cm) at a population density of onto silica gel (70–230 mesh size, 100 g) and eluted with 10 to 12 chicks per cage. The chicks were fed on chick 200 mL of the following solvents of increasing polarity: mash obtained from GAFCO, Tema, Ghana, and water petroleum ether, chloroform, ethyl acetate and metha- ad libitum. Temperature was kept at 29 °C, and over- nol to obtain 100 fractions. All fractions were analysed head incandescent illumination was maintained on a by thin layer chromatography (TLC), and samples with 12-h light–dark cycle. similar TLC profiles combined together. The fraction eluted with petroleum ether : ethyl acetate (6:4) yielded colourless prismatic crystalline solids that were purified Experimental design. Seven-day-old chicks were ran- by washing and recrystallization with ethyl acetate : pet- domly selected and put into groups of five animals each: ether mixtures. The recrystallization process was repeated the negative control group was receiving saline that was several times to obtain the pure compound (1.674 g). the vehicle for reconstituting the extracts and various doses (mg/kg) of the positive control and experimental drug treatment groups. The vehicle, extracts, and the Spectroscopic analysis. 1H–, 13C–, DEPT and two- isolated compound (3–90 mg/kg) were administered dimensional nuclear magnetic resonance [NMR; correla- orally, whereas the standard reference drug, diclofenac, tion spectroscopy (COSY), Nuclear Overhauser effect was administered intraperitoneally 1 h and 30 min prior spectroscopy (NOESY), heteronuclear multiple-quantum to carrageenan administration, respectively. All experi- correlation spectroscopy (HMQC) and heteronuclear mental protocols were in compliance with the National multiple-bond correlation spectroscopy (HMBC)] spectra Institute of Health guidelines for the care and use of lab- were obtained on a JEOL 500-MHz instrument (JEOL oratory animals and were approved by the Department Ltd, Welwyn Garden City, UK) essentially as described pre- of Pharmacology, Faculty of Pharmacy and Pharmaceu- viously (Roselli et al., 2012). AWaters Synapt G2 TOF mass tical Sciences, KNUST Ethics Committee. spectrometer (Waters, UK) with an electrospray ionization probe was used to acquire data over a mass range of 50–800 u (Bose et al., 2013). Antiinflammatory assay: carrageenan-induced foot oedema. The chick carrageenan-induced foot oedema model of inflammation described by Roach and Sufka (2003) was X-ray analysis. The molecule crystallizes in the mono- employed with some modification. Carrageenan (10 μLof clinic space group P21 with two molecules in the asymmet- a 1% suspension in saline) was injected sub-plantar into ric unit. The details of the data collection and important the right footpads of the chicks. The foot volume was features of the refinement are provided in Table S1. measured before injection and at hourly intervals for 5 h Single crystal X-ray diffraction data were collected on after injection by water displacement plethysmography as an Agilent Super Nova Dual Diffractometer