Antiinflammatory Properties of the Stem-Bark of Anopyxis Klaineana and Its Major Constituent, Methyl Angolensate

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 laboratories 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 (Agilent described by Fereidoni et al. (2000) using an electronic Technologies Inc, Santa Clara, CA) with Cu-Kα radiation Von Frey plethysmometer (Model 2888, IITC Life Science (λ =1.5418Ǻ) at 150 K. Data reductions were carried out Inc. Ca 91367 Canada). The oedema component of inflam- using CrysAlisPro V171.36.28. The structure was solved mation was quantified by measuring the difference in foot by direct methods and refined by full-matrix least squares volume before carrageenan injection and at an hourly time on the basis of F2 using SHELXL-97. All hydrogen atoms interval. were refined anisotropically while hydrogen atoms were treated using a riding model. The final refinement included 639 parameters for 9632 data and converged with Statistical analysis. The percentage difference from the r = 0.0301. The absolute stereochemistry was confirmed initial foot volumes at time zero values was averaged by refinement of the Flack parameter 0.05(4). for each treatment group, and the total foot volume for each treatment group calculated in arbitrary unit as Methyl angolensate. Colourless crystals; electrospray the area under the curve (AUC). The percentage ionization mass spectrometry (ESI-MS) m/z:[M+H]+

Copyright © 2014 John Wiley & Sons, Ltd. Phytother. Res. 28: 1855–1860 (2014) ANTIINFLAMMATORY PROPERTIES OF ANOPYXIS KLAINEANA 1857 at 471.2387 (calc 471. 2383) and analysed for C27H35O7; À1 [α]D = À40° (c 0.45); IR (KBr) νmax cm : 3080, 1745, 1720, 1618 and 875; UV (MeOH) λmax nm (ϵ): 209 (6000); 13C NMR (CDCl3): δ 13.7 (C-18), 21.0 (C-29), 21.6 (C-19), 23.7 (C-11), 25.7 (C-28), 29.2 (C-12), 32.6 (C-6), 33.7 (C-15), 39.3 (C-2), 41.3 (C-13), 42.8 (C-5), 43.9 (C-10), 48.0 (C-4), 49.8 (C-9), 52.0 (OMe), 77.1 (C-1), 79.5 (C-17), 80.1 (C-14), 109.9 (C-22), 111.5 (C-30), 120.8 (C-20), 140.7 (C-21), 142.1 (C-23), 145.7 (C-8), 170.0 (C-16), 173.8 (C-7), 212.7 (C-3). 1H NMR (CDCl3): 3.53 dd (6.3, 4.2 Hz, H-1), 2.52 dd (14.3, 4.2 Hz) and 2.95 m (H-2), 2.89 m (H-5), 2.60 m and 2.26 m (H-6), 2.19 m (H-9), 2.20 m and 1.60 m (H-11), 1.90 dt (14.1, 4.6 Hz) and 1.13 dd (13.4, 4.6 Hz, H-12), 2.95 m and 2,58 m (H-15), 5.66 s (H-17), 0.87 s (H-18), 0.95 s (H-19), 7.43 br t (0.84 Hz, H-21), 6.38 dd (1.9, 0.8 Hz, H-22), 7.38 t (1.7 Hz, H-23), 1.90 s (H-28), 1.25 s (H-29), 5.16 s and 4.90 s (H-30). Figure 1. Time-course oedema development following carrageenan injection into chick paws and dose-dependent antiinflammatory effect of diclofenac. All data for the treated groups are significantly different (p < 0.05) from the negative control values at each time RESULTS AND DISCUSSION point.

The stem-bark of A. klaineana has numerous ethnomedicinal uses including the treatment of inflammatory conditions associated with skin diseases and wound healing, malaria, pneumonia, bronchitis, skin infections and sexually transmitted diseases. Given that no scientific work has ever been presented for the plant, the present study investigated the antiinflammatory activities of A. klaineana stem-bark extracts along with the identification of the major active principle(s). The carrageenan-induced oedema model in animals is one of the most conventional methods of antiinflammatory activity evaluations of test compounds in vivo (Habtemariam, 2001). Injection of carrageenan into footpads of the chicks results in a time-dependent oedema formation that is attributed to the development of inflammatory condition (Roach and Sufka, 2003). This carrageenan- induced paw oedema resulted from a rapid release of acute phase mediators such as histamine and bradykinins followed by cyclooxygenase products including prosta- Figure 2. Total antiinflammatory activity of diclofenac during the glandins (Silva et al., 2005; Woode et al., 2008). As shown 5-h observation period. The dose-dependent effect of diclofenac in Fig. 1, the highest oedema formation under our exper- is shown from data obtained from the AUC as described in the Materials and Methods. Data are mean and SEM values (n = 5). imental condition was obtained during the first hour post- carrageenan injection, and the increased oedema was persistently evident during the whole 5-h observation Through a combination of column chromatography period. In order to validate the assay, a well known and recrystallization, the principal constituent of the antiinflammatory drug, diclofenac, was also included in methanol extract of stem-bark of A. klaineana was iso- our experiments. The dose-dependent and time-dependent lated as white crystalline compound. The ESI-MS anal- effect of diclofenac in suppressing the carrageenan- ysis in a positive ion mode using an accurate mass induced inflammation is shown in Fig. 1. The overall capable A Waters Synapt G2 TOF mass spectrometer antiinflammatory activity of test agents during the entire gave the molecular ion as [M + H]+ at 471.2387 (calc observation period can also be assessed from the AUC 471. 2383) and analysed for C27H35O7. Comprehensive analysis as demonstrated by the dose-dependent effect 1D (1H and 13C NMR, DEPT) and 2D NMR (COSY, of diclofenac (Fig. 2). On the basis of this validated assay, HMQC, HMBC, NOESY) analyses gave data consis- the antiinflammatory potential of three successive Soxhlet tent with the assignment of the compound as methyl extracts of stem-bark of A. klaineana was assessed. It is angolensate (1, Fig. 5). The spectroscopic data obtained apparent from Fig. 3 that the methanol extract, which for the compound in general were also in agreement was obtained with the highest yield, displayed the most with those published previously for 1 of various origins antiinflammatory effect. The fact that the methanol extract (Chan et al., 1967; Chiruvella et al., 2007). Given the displayed significant time-dependent antiinflammatory complex stereochemistry of the compound could not activity even at the lowest dose of 30 mg/kg (Fig. 4) appears be established solely from the NMR data, however, we to agree with the traditional uses of the plant as a popular have further undertaken an X-ray crystallography study. antiinflammatory agent. The crystalline methyl angolensate sample studied by

Figure 3. Comparison of antiinflammatory activity of methanol, ethyl acetate (EtOAc) and petroleum ether (petrol) extracts of Figure 6. Thermal ellipsoid plot of methyl angolensate with ellip- Anopyxis klaineana stem-bark. The total antiinflammatory activity soids drawn at 50% probability level (hydrogen atoms omitted during the 5-h observation period was calculated from the AUC as for clarity). described in the Materials and Methods. Data are mean and SEM values (n = 5). * indicate significant difference (p < 0.05) from other treatment groups of the same dose. To date, the tetranortriterpenoid methyl anglensate, classi- fied under the andirobins (Kipassa, 2008), has been isolated from several species of plants in the family Meliaceae. These include the stem-bark of Entandrophragma angolense (Akisanya et al., 1960), the wood of Khaya senegalensis (Abdelgaleil and Nakatani, 2003), the fruits of Swietenia mahagoni (Lin et al., 2009), the seeds of Carapa guianensis (da Silva et al., 2009) and Neobeguea mahafalensis (Naidoo et al., 2003), the heart wood of Cedrela odorata (Chan et al., 1967) and the callus cultures of Soymida febrifuga (Chiruvella et al., 2007). Limonoids occurrence is known to be confined to plants in the order of Rutales, in particular, members of the family Meliaceae, although a limited range of structures is found in Rutaceae, Cneoraceae, Ptaeroxylaceae and Simaroubaceae. The present study reports for the first time the occurrence of methyl angolensate from A. klaineana of the family Rhizophoraceae. The isolated compound was evaluated for its antiinflammatory activity at various doses and its effect compared with the positive control, diclofenac, through ED50 analysis. The results showed that methyl angolensate Figure 4. Time-dependent antiinflammatory effect of the most ac- has a potent antiinflammatory activity with an ED value tive stem-bark extract (methanol). All data are mean and SEM 50 values (n = 5). Except for one data point shown by asterisk, all of 4.05 ± 0.0034 mg/kg, which was less than twofold weaker data were significantly (p < 0.05) different from their perspective than diclofenac (2.49 ± 0.023, n = 5). In comparison to negative control group. diclofenac that was administered via the intraperitoneal route, methyl angolensate was administered orally and hence subjected to extensive degradation in the gut and liver prior to it being available within the circulatory sys- tem and induce its antiinflammatory effect. Hence, the observed antiinflammatory effect, although slightly weaker than the positive control, is a significant finding. In the study by Penido et al. (2005), the antiinflammatory effects of six combined natural tetranortriterpenoids including 6% of methyl angolensate of C. guianensis seed origin was evaluated using the zymosan-induced arthritis Figure 5. Structure of methyl angolensate (1). method in mice. The result indicated that the six tetranortriterpenoids through a synergistic effect pro- duced a significant antiinflammatory effect that attri- X-ray was characterized as a new polymorphic form of butes to the pharmacological effects observed for the that reported previously (Sanni et al., 1987). The essen- seed oil (Penido et al., 2006). In another study, Thioune tial details on the molecular structure (Fig. 6) however et al. (1999) investigated the antiinflammatory activity are closely similar, and the overall data are provided in of the aqueous and nonpolar extracts of the stem-bark Table S1. of K. senegalensis and suggested that the observed

Copyright © 2014 John Wiley & Sons, Ltd. Phytother. Res. 28: 1855–1860 (2014) ANTIINFLAMMATORY PROPERTIES OF ANOPYXIS KLAINEANA 1859 antiinflammatory effect may be due to the stem-bark In conclusion, the results of this study have clearly constituents, methyl angolensate (1) and methyl demonstrated significant antiinflammatory activity of hydroxyangolensate (Thioune et al., 1999). In an in vitro the stem-bark of A. klaineana. The finding therefore jus- study by Ferraris et al. (2011), methyl angolensate (1) tifies to some extent the folkloric use of the stem-bark of from C. guianensis seeds has also been shown to suppress A. klaineana for treating inflammatory conditions asso- the adhesion of eosinophils to tumour necrosis factor-α- ciated with skin conditions, joint aches and would activated endothelial cells. The antiinflammatory activity healings. The occurrence of the tetranortriterpenoid observed for 1 in the present study was thus in agreement methyl angolensate (1) in the plant as well as in the fam- with previous studies and provides the first direct evi- ily Rhizophoraceae is reported now for the first time. dence on the antiinflammatory potential of the com- Our data also show that the antiinflammatory activity pound. Because TLC analysis of the second most active of the stem-bark of the plant in part could be due to extract, the ethyl acetate, has revealed methyl angolensate its major constituent, methyl angolensate (1). as its principal constituent (data not shown), the compound should be regarded as the major antiinflammatory principle of the plant. The existence of other minor com- Acknowledgements ponents in A. klaineana either being more potent than 1 or those working through synergism with 1 however We are thankful to Mr Kofi Asare and Mr Kwaku Tawiah Agyare, the remains to be established. Other biological activities herbalists who assisted in the processing and identification of the plant material. We also wish to thank the technical staff of the University of reported for 1 include antimicrobial (Chiruvella et al., Greenwich NMR and MS laboratories. 2007), spasmolytic (Orisadipe et al., 2001), antiulcer (Njar et al., 1995), anticancer (Chiruvella et al., 2008), antiallergic (Penido et al., 2005), insect antifeedant activ- Conflict of Interest ity (Abdelgaleil and Nakatani, 2003) as well as behavioural and sedative effects in rodents (Amos et al., 2002). The authors declared that there is no conflict of interest.

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