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Cardiovascular Effects of the Alkaloid Hippadine on the Isolated Perfused Rat Heart

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Cardiovascular Effects of the Alkaloid Hippadine on the Isolated Perfused Rat Heart Int. J. Med. Arom. Plants, ISSN 2249 – 4340 RESEARCH ARTICLE Vol. 2, No. 1, pp. 172-177, March 2012 Cardiovascular effects of the alkaloid hippadine on the isolated perfused rat heart P. MUGABO1, K.C. OBIKEZE1*, A. NJAGI1, A.P. BURGER2, I. GREEN3, D.D. DIETRICH2 1Department of Pharmacology, University of the Western Cape, Bellville, South Africa 2Department of Physiology, University of the Western Cape, Bellville, South Africa 3Department of Chemistry, University of the Western Cape, Bellville, South Africa *Corresponding Author, Tel: +27 21 959 3665, Fax: +27 21 959 3407 Article History: Received 29th January 2012, Revised 20th February 2012, Accepted 20th February 2012. Abstract: Crinum macowanii has been used extensively in traditional medicines for treatment of various illnesses such as oedema and ‘heart disease’. Previous studies of the crude bulb extracts on Langendorff perfused isolated rat hearts indicated a positive inotropic effect. The aim of this study was to isolate and characterize compound(s) from C. macowa- nii with cardiovascular effects similar to that observed with the crude extracts of the plant. The methanol extract of dried bulbs was extracted for alkaloids, and structural elucidation of the isolated alkaloid identified it as hippadine. The cardi- ovascular effects of hippadine was evaluated in vitro in isolated perfused rat hearts using the “double sided” working heart system. Perfusion with 0.5 μg/ml and 5.0 μg/ml hippadine in Krebs-Hanseleit buffer led to significant decreases in coronary flow, aortic output, cardiac output, systolic pressure, and heart rate, accompanied by increases in diastolic pres- sure. Hippadine exhibited a negative chronotropic and inotropic effect on the isolated rat heart and is responsible either partly or fully for the cardiovascular effects of C. macowanii. Keywords: Alkaloid; Cardiovascular; Crinum macowanii; Hippadine; Rats. Introduction 2010; Jayakumar and Sheu 2011; Jensen et al. Plants in the Amaryllidaceae family have 2011; Nair et al. 2011). As such it was hypothe- been used quite extensively in traditional medi- sized that the cardiovascular effects produced by cines for the treatment of various illnesses. Cri- the plant extracts were partly or wholly due to num macowanii, a member of this family of alkaloids present in the plant. The aim of the plants has found extensive use in traditional present study was to isolate and characterise a medicines for the treatment of various illnesses cardioactive alkaloid from extracts of the bulbs of C macowanii. such as oedema, ‘heart disease’, rheumatic fev- er, cancer and skin diseases (Duncan et al. 1999; Van Wyk et al. 2000; Elgorashi et al. 2001; El- Materials and Methods gorashi et al. 2002; Elgorashi et al. 2003). In a previous study to examine the cardiovascular Isolation of hippadine effect of the plant, Mugabo et al. (2001) re- Crinum macowanii was obtained from ported a positive inotropic effect and no effect Kirstenbosch botanical gardens (Cape Town, on heart rate in isolated male Wistar rat hearts South Africa), identified and a voucher speci- perfused with the crude extract of the bulbs of men was deposited at the herbarium at the Uni- Crinum macowanii via a Langendorff perfusion versity of the Western Cape (UWC). Fresh system. Plants in the Amaryllidaceae family are bulbs were diced, dried at 30˚C to a constant rich in alkaloids and many alkaloids extracted mass and milled to a fine powder. The pow- from plants have been shown to exhibit cardi- dered bulbs were extracted with methanol, and ovascular activity (Andraws et al. 2005; Osorio the methanol extract extracted for alkaloids ac- et al. 2010; Rostoff et al. 2010; Wang et al. cording to method described by Nair et al. *Corresponding author: (E-mail) kobikeze<A.T.>uwc.ac.za http://www.openaccessscience.com ©2012 Open Access Science Research Publisher [email protected] 173 Int. J. Med. Arom. Plants Cardiovascular effects of hippadine in rat hearts (2000). The alkaloid-containing extract was tinct working heart systems via a system of taps fractionated by column chromatography (CC) to reduce the risk of cross-contamination of the using a silica gel (70-230 mesh) column and perfused drug/extracts. The rats were anaesthe- ethyl acetate: hexane (1:4); (2:3); (3:2); and tized with sodium pentobarbitone (30 mg/kg (4:1) as sequential eluents. Fraction G from the IP), and the beating hearts were rapidly excised ethyl acetate: hexane (1:4) elution showed sig- and immersed in cold (<4oC) Krebs-Hanseleit nificant cardiovascular effects in vivo (results buffer. The excised hearts were then mounted not shown) and was subjected to further CC and cannulated for working heart perfusion ac- separation using the eluents mentioned above. A cording to the method described by Sutherland pure compound C045 was isolated from fraction and Hearse (2000a,b), and then perfused retro- E of the ethyl acetate: hexane (2:3) eluent and gradely (Langendorff perfusion) for ten minutes purified by crystallization from chloroform. with Krebs-Hanseleit buffer, following which a Identification tests revealed that C045 was hip- ten minute working heart perfusion with either padine, an alkaloid previously isolated from the control (Krebs-Hanseleit buffer or adrenaline) Amaryllidaceae family (Hutchings and Meyers or test substance (hippadine) occurred. Blood 1996; Torres et al. 2004). pressure (BP) and heart rate (HR) were meas- ured via a pressure transducer, connected to a side arm of the aortic cannula and linked to a Animals computer system running the chart recorder Normotensive male Wistar rats weighing (version 2.0 Gentronics, SA). Coronary flow 250 - 350 g and less than four months old were (Qe) was measured as runoff from the organ used for the experiments. The animals were ob- chamber per minute, aortic output (Qa) as runoff tained from the Physiology department, UWC from the cardiac output compliance chamber per and were allowed free access to food and water. minute, and cardiac output (CO) measured as Ethical approval for the study was obtained and the sum or aortic output (Qa) and coronary flow all animals were treated according to ethical (Qe). The effects of the standard drug (adrena- regulations as required by the University of line - 0.01 μg/ml and 1.0 μg/ml) and test sub- Western Cape ethics committee. stance (hippadine - 0.5 μg/ml and 5.0 μg/ml) on these cardiovascular parameters following working heart perfusion with the following per- Drugs and Chemicals fusion protocol were evaluated: Adrenaline (Bodene; Cape Town, South i) 0 – 10 minutes (reperfusion period): Langen- Africa) and hippadine were dissolved in normal dorff perfusion with KHB. saline and further diluted with Krebs-Hanseleit ii) 10 – 20 minutes (stabilization period): Work- buffer (KHB) solution. Fresh KHB solution ing heart perfusion with KHB. containing (in mM); 118.5 NaCl, 25.0 NaHCO3, iii) 20 30 minutes (drug perfusion period): 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2 – and 10.0 g glucose (all Sigma; Cape Town, Working heart perfusion with drug in KHB. South Africa) was prepared daily (Sutherland iv) 30 – 40 minutes (stabilization period): Same and Hearse 2000a). as ii above. v) 40-50 minutes (drug perfusion period): Same Measurement of blood pressure and heart rate as iii above. in vitro vi) 50-60 minutes (stabilization period): Same A modified version of the isolated perfused as ii above. working heart model was used to evaluate the in vitro effects of hippadine (Depre 1998; Suther- Statistical analysis land and Hearse 2000a,b). The modification as described by Mugabo et al. (2001) was to enable The data is expressed as mean ± SD of HR, the perfusion of the working heart from two dis- SP, DP, Qe, CO and Qa for five (5) replicates. http://www.openaccessscience.com Mugabo et al. [email protected] 174 Int. J. Med. Arom. Plants Cardiovascular effects of hippadine in rat hearts Statistical significance between means for con- site to that observed with adrenaline perfusion trol and test substances was calculated using the (Figure 2). unpaired Student’s t-test (p<0.05). 10 * 5 * 0 Results 0.5 5.0 0.01 1.0 -5 Hippadine Re-crystallization yielded white crystals -10 Adrenaline with the chemical formula C16H9NO3. Mp 217– -15 * o 218 C. HREIMS m/z value 263.247289. A -20 search of chemical database Chembase® and -25 other literature identified the isolated compound %Change Heart in rtate -30 as hippadine (Hutchings and Meyers 1996; -35 Torres et al. 2004). -40 * In isolated perfused working hearts, perfu- Figure 2: Effect of hippadine (0.5 μg/ml and 5.0 sion with hippadine (0.5 μg/ml and 5.0 μg/ml) μg/ml) and adrenaline (0.01 μg/ml and 1.0 produced significant dose dependent decreases μg/ml) on heart rate in isolated perfused work- in systolic pressure (10.03% ± 0.012 and ing rat hearts. 20.56% ± 0.021 for 0.5 μg/ml and 5.0 μg/ml * P <0.05 with respect to control (Krebs- doses respectively). This effect was the opposite Hanseleit buffer). of the significant dose dependent increases in systolic pressure produced by adrenaline (0.01 μg/ml and 1.0 μg/ml) (Figure 1). Diastolic pres- Hippadine perfusion led to dose-dependent sure increased in a dose-dependent manner with statistically significant decreases in coronary hippadine perfusion, although statistically sig- flow (15.82% ± 0.109 and 48.83% ± 0.172 for nificant only with the 0.5 μg/ml dose. This ef- 0.5 μg/ml and 5.0 μg/ml doses respectively) and fect was opposite to that obtained for adrenaline cardiac output (14.35% ± 0.670 and 89.13% ± (Figure 1). 0.390 for 0.5 μg/ml and 5.0 μg/ml doses respec- tively) (Figures 3-4).
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