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320 MANSURAH A. ABDULAZEEZ et al.antithrombogenic properties (6). In the cardiovascularsystem, NO is released from the endotheliumin response to a physiologic stimulus to activateguanylate cyclase in vascular smooth muscle cellsand increase cGMP levels to mediate relaxation(7). The interruption of NO synthesis with severalL-arginine analogues such as N G -nitro-L-argininemethyl ester (L-NAME) has been shown to inducevasoconstriction and arterial hypertension inexperimental animals (8) making it a suitablemodel for studying the antihypertensive effects ofnew drugs.Angiotensin converting enzyme (ACE) is atransmembrane zinc metallopeptidase, involved inthe regulation of vascular tone by convertingangiotensin I (an inactive decapeptide) toangiotensin II (a potent vasoconstrictor). Theenzyme also inactivates the vasodilatory nonapeptidebradykinin (9). The ACE inhibitors are used forthe treatment of hypertension and prevention ofchronic heart failure (10). The effect of ACEinhibitors on the NO/cGMP pathway is not wellestablished (11-13), even though it is well knownthat the antihypertensive effect of these drugs isattributed to decreased angiotensin II and increasedvasodilatory kinins and NO (6). Due to the highincidence and morbidity of hypertension, variousdrugs and regimes have been advocated for its control.Most of these drugs do not possess completecurative properties; and some demonstrate betterefficacy but possess side-effects (14). Recently,attention has been drawn towards herbal sourceswhich are used traditionally as potential therapeuticagents in the prevention and management of cardiovasculardiseases (15, 16).Peristrophe bicalyculata (Retz) Nees is nativeto warm tropical regions of Africa, in the Sahel partof the region from Mauritania to Niger and NorthernNigeria, India, Burma and Thailand (17). The leavesof the plant have analgesic, antipyretic, anti-inflammatoryactivities, and antibacterial, fungistatic andbacteriostatic properties (17, 18). Studies havedemonstrated the anticancer activity of oils fromPeristrophe bicalyculata using MCF-7 (humanbreast tumor) and MDA-MB-468 (human breasttumor) cells (19). The anticancer activity of crudeextracts of the plant against Ehrlich ascites carcinoma(EAC) cell lines (20) and human mouth epidermalcarcinoma (KB) cells (21) have also beenreported. In South-West Nigeria, the plant is used asgreen manure as well as in the treatment of hypertensionand cardiovascular-related diseases (22).The present study was aimed at partially purifyingthe antihypertensive extract of Peristrophe bicalyculata,determine the ACE inhibitory activities of thefractions and identify the antihypertensive component(s)/constituent(s)present.MATERIALS AND METHODSChemicals and reagentsHippuryl-histidyl-leucine (HHL), angiotensinconverting enzyme (ACE), N G -nitro-L-argininemethyl ester (L-NAME) and captopril were obtainedfrom Sigma Chemical Co. (St. Louis, MO, USA).All other chemicals were of analytical grade.Preparation of plant materialThe leaves of Peristrophe bicalyculata (Retz)Nees were harvested at maturity in the month ofJune, 2010 at Ibadan, Oyo State, Nigeria. The plantwas identified and authenticated by the botanist inthe herbarium of the Department of BiologicalScience, Ahmadu Bello University (ABU), Zaria,with a voucher number 2863.The leaves of the plant were washed cleanunder running water, air-dried in the laboratory andpowdered. The powdered plant (500 g) was defattedin n-hexane before extracting with methanol. Themethanol extract was dissolved in distilled waterand then partitioned in ethyl acetate and n-butanolusing separating funnel, to obtain ethyl acetate fractionof methanol extract, butanol fraction ofmethanol extract and water fraction of methanolextract. The cold and hot water extracts wereobtained by stirring (Harmony Hot Plate Stirrer,Japan) in cold and hot water, respectively, sievedusing a muslin cloth and then filtered under suctionpressure with Whatmanís filter paper (No. 1). Allextracts were then concentrated under reduced pressureusing a rotary evaporator (Buchi, Switzerland),lyophilized (Christ Alpha 1-2 LD, Germany) andstored at 4 O C until needed.Bioassay-guided fractionation of cold waterextract of Peristrophe bicalyculataThe cold water extract of Peristrophe bicalyculata,was partially purified by column chromatographyafter thin layer chromatographic separation. Thethin layer chromatography was carried out usingethyl acetate : formic acid : methanol (1 : 1 : 3,v/v/v) solvent which gave five separate constituents,visible under UV-spectrum. Column chromatographyusing silica gel was then run with ethyl acetate(100%), ethyl acetate : formic acid : methanol (15 :2 : 0.5, v/v/v), ethyl acetate : methanol (1 : 1, v/v),ethyl acetate : methanol (1 : 3, v/v), ethyl acetate :methanol : formic acid (1 : 3 : 1, v/v/v) and methanol
Bioassay-guided fractionation and antihypertensive properties of... 321: formic acid (9 : 1, v/v). Elution and fractionationwere controlled by TLC and similar fractions werecombined.Determination of angiotensin-converting enzymeinhibitory activityThe assay for ACE inhibitory activity wasdetermined using the Cushman and Cheung (23)method with some modifications on the assay conditions.Briefly, the inhibitor solution (purifiedextract) was added to 0.1 M potassium phosphatebuffer (pH 8.3), which consists of 5 mM hippurylhistidyl-leucine(HHL), 0.1 M potassium phosphateand 0.3 M NaCl (pH 8.3). Then, the enzyme,ACE was added to the mixture and incubated at37 O C for 30 min. The reaction was terminated byadding 0.25 mL of 1 M HCl, and then 1.5 mL ofethyl acetate was added to extract the hippuric acidformed by the action of ACE. The ethyl acetatewas removed by heat evaporation, residual hippuricacid (HA) dissolved in 1 mL of deionizedwater, and absorbance of the solution was measuredat 228 nm to determine the hippuric acid concentrationand ACE inhibitory activity. The concentrationof ACE inhibitor required to inhibit 50%of the ACE activity under the above assay conditionswas defined as IC 50 .Experimental designAll experimental protocols were approved andconducted with strict adherence to guidelines andprocedures of the Institutional Animal Care and UseCommittee of the Natural Product Research andDevelopment Centre, Faculty of Pharmacy, ChiangMai University, Thailand. One hundred apparentlyhealthy male Sprague-Dawley (SD) rats of abouteight weeksí old, weighing between 180 and 230 gwere obtained from the National Laboratory AnimalCentre, Mahidol University, Salaya, NakhonPathom, Thailand, and maintained in an air-conditionedanimal house of the Faculty of Pharmacy,Chiang Mai University, Thailand. They were fednormal rat chow and allowed access to clean waterad libitum. The rats were allowed to acclimatize fortwo weeks before the commencement of the experiment.The experiment was carried out in two phases.In the first phase, rats were divided into 4 groups of6 rats each: Group 1 (control) - rats were intravenouslygiven normal saline, groups 2, 3 and 4were intravenously injected with a solution of L-NAME (3 mg/kg) until blood pressure reached maximum.Captopril (5 mg/kg), water fraction ofmethanol extract (10 mg/kg) and cold water extract(10 mg/kg) were intravenously administered tohypertensive rats in groups 2, 3 and 4, respectively.In the second phase of the experiment; rats weredivided into 6 groups of 6 rats each: Group 1 ratswere intravenously given normal saline, groups 2, 3,4, 5 and 6 were intravenously injected with a solutionof L-NAME (3 mg/kg) until blood pressurereached maximum. Fractions 1, 2, 3, 4 and 5obtained after partial purification of the cold waterextract, were given to rats in second, third, fourth,fifth and sixth groups, respectively.Acute toxicity studiesAcute oral toxicity studies of extracts ofPeristrophe bicalyculata were carried out asdescribed by Lorke (24) with oral administration ofincreasing doses of the extracts from 10 mg/kg to5000 mg/kg, while animals were observed forbehavioral changes, toxicity and mortality for 24 h.Blood pressure and heart rate measurementRats were fixed on a supine position on a dissectingtable and anesthetized by intraperitonealadministration of pentobarbital sodium (40mg/kg). A longitudinal mid-tracheal incision,approximately 2 cm long was made in order toexpose the trachea, the right jugular vein and bothcommon carotid arteries. The trachea was cannulatedwith a polyethylene tube (2.75 mm diameter)to maintain a free airway, while the right jugularvein was cannulated for the administration ofextracts and isotonic saline solution. The systemicblood pressure was recorded from the cannulatedleft carotid artery, connected to a physiologicalpressure transducer (Statham P23 AC Strain gaugeTansducer, Laboratories, Inc. Hato Rey, PuertoRico) and displayed on a Grass 7D Polygraph(Grass Instrument Co., Quincy, Mass, USA). Thecannulation of the carotid artery was performed inthe same manner as that of the jugular vein, andthe polyethylene tube (1 mm diameter) filled withheparin sodium in saline solution was used. Bloodpressure and heart rate were monitored untilsteady base-line levels were obtained. L-NAME at3 mg/kg was administered via venous cannula toinduce hypertension. Blood pressure was allowedto stabilize for 30 min before administration ofextracts and fractions through the jugular veincannula. Changes in blood pressure were recognizedas difference between the steady state valuesbefore and the peak readings after injection (25).Mean arterial blood pressure (MABP) was calculatedas the diastolic BP plus one-third of the pulsewidth (systolic BP - diastolic BP).
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PHARMACEUTICAL TECHNOLOGY347. £uka
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