Intemational Journal of Pharmacology 4 (6): 487-491, 2008 ISSN 1811-7775 © 2008 Asian Network for Seientifie Information ‘The Effect of Ethanol Extract of Kucai (Allium schoenoprasum L.) Bulbs on Serum Nitric Oxide Level in Male Wistar Rats AL, Amalia, 'E.Y. Sukandar, *R M.A. Roesli and "LL Sigit °Pharmacology-Clinical Pharmacy Rescarch Group, School of Pharmacy, Institut Teknologi Bandung, JI. Ganesa No, 10, Bandung, Indonesia "Department of Intemal Medicine, Padjadjaran University, Hasan Sadikin Hospital, J Pasteur No, 38, Bandung, Indonesia Abstract: The aim of ths study was to determine the elect of ethanol extract of Kucai bulbs on serum nitiic oxide (NO) levels in rts, in an attempt to study the mechanism of antihypertensive action. The measurement ‘of NO level was conducted by Griess colorimetric assay. The male Wistar rats were divided randomly into three groups; the first group was treated with kucai extract of 100 mg ke body weight (kuca-treated group): the second group with isosoride dinitrate (ISDN) of 0.18 mg kg" body weight (ISDN-treated group) and the third group with vehicle only (control group). The results showed that at 30:min (Ty) the mean NO level in kucai- treated group increased significantly (138.69.-169 38%) compared to baseline (,); similarly, in ISDN-treated group, the mean NO level increased (212.08+140 01%) significantly. At To, average NO levels in kucaitreated ‘1 0up was significantly higher than those in control group (p= 0.018), while those in ISDN-treated group were ‘ot signifiantly higher than those in control group (p = 0.436), Compared to kuea-treated group, NO Tevels in ISDN-reated group at Ts was significantly different (p ~ 028), From this study it can be concluded that cthanol extract of kucai bulls increases significantly serum NO level in experimental rats. Therefore, itis preatsumed that antihypertensive effect shown by fucai is, one of which, due to mechanism of vasodilator action, The present results showved that kuca’ increased NO levels and this might be one mechanism of blood pressure lowering action of kuca Key words: Allium schoenoprasum I... kucai, nitric oxide, vasodilator, high blood pressure INTRODUCTION Nitric exide (NO) is a chemical compound that plays 8 significant role in pathogenesis of several diseases, including hypertension. A physiological fimetion of NO. was discovered in the vasculature when it was shown that the endothelium-derived relaxing factor (EDRF) described by Furchgott and Zawadeki in 1980 is accounted for by formation of NO by endothelial cells (Furehgott, 1983; Sun ef al, 2003), NO is produced by endothelium cell or nitsate group of digs, is the endogenous activator of soluble guanylate cyclase and plays mainly in this mechanism, NO is biosynthesized from L-arginine amino acid by at least three different isoform types of NO. synthase, that is, neuronal NO synthase (nNOS), endotelial NO synthase (eNOS) and inducible NO) synthase (NOS) (Balbatun e7 af, 2003, Gharavi and ELKadi, 2003), Free-tadical NO produced will immediately react with molecular oxygen ancl water to form a variety of end-products, including nitrite (NO>), nitrate (NO,~) and Sxuitrosothiol (RENO) (Gharavi and ElKadi, 2008; Brovkovych ef al, 1999). Free-radical NO is promplly ‘ngotivated by superoxide, Fe(II) and Fe(I), that are all in significant amount in biological system. The endothelium produced NO will diffuse into smooth muscle and activate Busianylate cyclase enzyme (atrial natriuretic peptide receptor 4), This enzyme stimulates cyclic guanosine 3¥,S-monophosphate (COMP) synthesis and then activates a number of protein kinase-dependent phosphorilations in smooth muscles and eventually produces dephosphorilation of myosin chain of the smooth muscles (Fig, 1), Caleiurn ion release will further tigger smooth muscle cell relaxation, reducing peripheral resistance, hence vasodilation occurs (Katzung and Chatterjee, 2007), By vasodilation the blood presure will decrease. Kucai (Allium schoenoprasum, L.) is @ medicinal plants that has been used traditionally in Indonesia for lowering blood pressure. Kucai postesses a good prospect to be developed ax phytopharmaceutical Corresponding Author: Lis Amalia, Pharmacology-Clinieal Pharmacy Research Group, School of Phasmaey, Institut Teknologi Bandung, J], Ganesa No. 10, Bandung, Indonesia ‘Tel: +0222-2509172/3M15 Fax: +6222-2504852 487Int. aed ow Nosdepenent stewing forces vesodliaas Inflemmation ' Fig. Biosinthesize of nitric oxide (adapted from Cardiovascular Physiology Concepts, Klabunde, RE), NO is biosyuthesized from L-arginine amino acid in endothelial cell by at least three different isoform types of NO synthase, that is, neuronal NO synthase (nNOS), endotelial NO. synthase (ENOS) and inducible NO synthase (INOS). Free- radical NO produced will activating guaianylate cycle enzyme, therefore stimulating GMP synthesis and then activates a number of protein kinase-dependent phosphorilations in smooth muscles and eventually produces dephosphorilation of myosin chain of the smooth muscles, Caleium ion release will further make smocth muscle cel relasation, reducing peripheral resistance, therefore vasodilation occurs product, so that it is expected as an altemative medication for hypertomion. For this purpose, various steps of testing through both preclinical and clinical tals must be performed to study the efficacy and safety of kucai at antihypertensive agents. This study was conducted to determine the mechanism of huca? action on vascular relaxation (vasodilation) in experimental animals. There re several methods of such mechanism, one of them is by measuring the NO level in animal serum. NO is produced by various types of cells in picomolar to nanomolar ammount and is half-life (T,) is very shor, ie. $ seein biological fluid, Therefor, iis very difficult to measure NO products directly; instead, analysis of nitrate (NO,-) and nitte (NO,") are wed to estimate NO level in biological fluid and call culture medium due to thei stability Several metbods have been developed in assessment of NO level by nitite measurement; ie, Gries colorimetric assey, chemilluinescence analysis, gas chromatograic analysis, hemoglabin-apping and fuorcmetrc method. Apart fiom that, there ae also ion-exchange, reverse- Pharmacol, 4(6): 487-401, 2008 phase ion pairing, reverse-phase high-performance liquid chromstography method developed to measure nitrite and nitrate in biological system, using absorbance of UV- visible, conductivity, electrochemical or fluorescence: (Gharavi and EI-Kadi, 2003), In this study, determination of NO level was performed through measurement by Griese colorimetric assay. This method is considered adequately simple due to itis wide used to analyze some amount of biological samples, including plasma (serum), urine, cerebrospinal fluid, saliva and cell culture media {Sum eral, 2003), This study would determine NO level of at serum as one of the methods to establish the mechanism of vasodilator action of kucai. In a earlier study on ethanol extract from kuca bulbs, an active ingredient presumed having antihypertensive effect had been identified as tetramethyloxamide (Fidrianny et al 2001), For this reason, this study used ethanol extract from fresh kucai bulbs, MATERIALS AND METHODS: ‘This smdy was performed in Pharmacology Leboratory-Schiool of Pharmacy Insititut Teknologi Bandung and Prodia Laboratory, Bandung-Indonesia at January 2008. ‘Determination of nitric oxide level was conducted by Griess colorimetric method In colorimettie Griess method, nitrite was first treated with diezotizing reagent e.g. sulfanilamide (SA) in acidic media to form a transient diazonium salt. This intermediate is then allowed to react with N-napithylethylenediamine (NED) to form a stable ‘azo compound, The intense purple calor of the product allows nitrite assay and can be used to measured nitrite concentration as low as ~0.5 IM. The absorbance of this adduct at S40 nm is linearly proportional to nitrite concentration in the sample, Preparation of kucai extract: Fresh kucai bulbs was purchased from local grocery market at Andir in Banchag, West Java, identified by botanists in the Herbarium Bandungense-School of Life Science and Technology, Institut Telmologi Bandung-Indonesia, The kucai extract was prepared with maseration-percolation method with 959% ethanol solutions. The extract then was vaporized with rotavapor until a concentrated extract was obtained Chemicals Griess reagent: Griess reagent was purchased from Oxford Biomedical Research, USA (Product #NB 88), Griess reagent consists of granulated cadmium (25 cadmium beads), ZnSO, solution (2- mL of 30% (wiv)Int. 2n80,), microcentifuge tubes (501.5 mL microventrfige tubes}, Color Reagent No. I (7 ml. sulfanilamide dissolved in 3N HCD, Color Reagent No. 2 (7 mL. N-{1-naphtyl] ethylenediamine dihydrochloride dissolved in deionized HO), nitrite standard (1.5 mL 500 pmol uL-' NaNO, equivalent to 500 4M NO), microtiter plate (One 96 well low protein binding plate with flat-bottom well), microtiter plate template (one printed template for duplicate assay), reagent reservoirs (three plastic troughs for dispensing and pipetting reagents), cadmium bead washing solutions (125 mL of 0.1 MHCI and 125 mL. of 0.1 MNH,OH), Drugs: Isosorbide dinitrate (ISDN) tablets, produced by PT. Kimia Farma Tbk. (Bandung, Indonesia) was used in this stody. ISDN was prepared as aqueous auspension and diluted with distilled water Animals: Fifteen male Wistar rat (weighing 250 to 300 g) were randomly selected and divided into three groups. The first group received a dove of 100 mg kg" of body weight of kucai, soeond group with a dose of (0.18 mg ka body weight of isosrbide dinitrate (SDN) and third group received only vehicle. Bloed samples were taken before treatment and 10,30, 60 and 90 min after the treatment (Sumino ef al, 2000). This study was conhieted in accordance with the Guideline for Care and Ue of Animals Laboratery of School of Pharmacy Instiat Teknologi Bandung, Samples preparation: Blood sample was incubated at room femperature for 45 min. then centifuged at 3000 rpm. for 15 min The serum was stored in 0.3 ml. tube and kept at -20°C until use, 10 to 50 pL, of dafrozen serum was. adjusted to 190 yL with distilled water and then added ith 10, of 30% (w/v) ZnSO, solution to make the total Volume 200 iL. Final solution was then mixed by vortexing, incubated for 15 min at room temperature and centriaged at 3,000-4,000 epra for $ min, The resulting supernatant was wansferred to microcentrifuge wabes containing 0.5 g gramslated cadmium (about 6-7 beads) and incubated at room temperature overnight with agitation. The sample solution was transferred to clean microcentrifuge tube, recentrifuge and the supematant was ested for NO, content, Standard solutions preparation: Standard solution was prepared by diluting nitrite standatd containing 1. mL. 500 pmol ul.~' NaNO, (equivalent to $00 pM NO). From the ‘solution, serial nivite standard solutions were prepared with NO concentration of 100, 50, 25,10, 51 and 1.5 4M (added with deionised water), Calibration curve ths made was uted to determine nitrite levels, Pharmacol, 4(6): 487-401, 2008 Procedure: Cadmium beads were washed in their individual mierotubes with H,O (21 mL}, 1 M HCL (21 mL) and0.1 MNH.OH, pH19.6(2-1 mL before used Standards and samples were prepared as described before. Samples were added to wells in duplicate, 20-100 mL, depending on the NO concentration in semple, distilled water was added to make a total volume of 100 mL. Fifty milliliter Color Reagent No. 1 was added followed by brief shaking. Fifty milliliter Color Reagent No. 2 was then added and shaken for 5 min at room temperature, Absorbance was read at $40 nm in microtiter plate reader. The standard curve was plotted and the sample concentration was estimated from the curve Statistical analysis: Statistical analysis was earried out by 2-way analysis of variance (ANOVA) by post-hoc test. Data are shown as mean valuesSD (standard deviation), A value of p=0,05 was considered statistically significant RESULTS AND DISCUSSION Isosorbide dinitrate (ISDN) have been widely used as vasosilators to teat acute myocardial ischemia, their biological effects being due to the release of NO. ISDN have longer terminal elimination half life than other nitovasodilator (20, intravenously, 64min sublingual) and has a high fst pass metabolism, broken down. to. Sisosorbide-mononitrate and. 2-isosorbide- smononitrate which are both more ative than their parent compound, ISDN has been used as a crug standard in several studies on determination of nitric oxide in both in vivo and in vitro systems (Paleiogin et al, 1998; Baluchmejadmojarad eta, 2003) ‘The done of extract used in tis study was at dase of 100 mg, kg body weight. This dose was chosen on the basis ofa earlier study suggesting that the kueat dose to have antihypertemive effect was 100 mg kg". While the dose of ISDN ie 0.18 mg kg body weight was derived fiom conversion of human therapeutic dose ‘The level of NO among the control, kucai-reated and ISDN-reated groups were not signficanily different, Up to 10 min (T,.) NO levels in kucabtreated, ISDNetreated and control aroups were decreased compared to those of| baseline (T;), but the decrease was not significant. After 10 min, NO levels increased in ISDN-treated and kucai- tueated groupe to their peaks (Table 1. Fig 2). ‘At Ty, mean NO levels of hucaitreated group increased sigificanly (%6 changes were 138,69160.38) compared to T, (p= 0.04), simiany, shat of ISDN-teated group increased significantly (@% changes were 21208414001, p = 0026), while in control group the increase was not significant (% changes wereInt. J. Pharmacol, 4 (0): 487-491, 2008 ‘Table: Sem Nivic Oxide (NO) eels change before and fer adnan of anal extract of Kiel Cetra op ‘Kira 100e ke" (B= 5) ISDN 018 maker a=5) ime oun, 2 NoUND Rowan, w 2a 90198 95 21506870 18 7108 Tm 1977348350 a1a9077 52 13. 99419.64, Ts 20078612211 ase 4623212 oe 1865 99 foes Te 16893:28.60 aur 43608815695 one rn ssemais owe ISDN = Tevorbode dniate, "Compared to Ty "Campard to coral gop, a vale of p00 wx onside sagt siglicnt. ATg NO kvel treated arp compara SDN exe aoip p= OS an Ty p= 0038 “Tuble2: Pecenage binges ofan NO eves at TT between direst ere gre eve Noda Tels Group Changes oie eas 100m 1386910038 bass svg a12081001 asm ISDN = Isosorbide daze « Conpared tocol grup, Camparedia ISDN-pestel grup, a value of prOC® as censdeed statically Sigisiean. Chanus were cused coapariag to baselpe (sun NO lesa “able 3: Company of sun NO levee anges bce dies sp Ta ToT Sou _ANDievlsh p _ANOleseiad p Centar 4172855 9D g7HN 3) Kiva! 10Omeke" 26166123859 asi 2212612064 007 ni ns ISDNCIemg he" ser 9TEI2 No ayeTONES7 O31 ISDN =i dintrate, Canpared to ISDN. weed gre, "Compared to cet go No level (a) 0 mm mw Tine (nin) 10 Fig, 2: Effect of ethanol extract of kuvcai administration to NO serum level, Male Wistar rats divided randomly into three groups: control group (#), ‘oucai-treated group (W) and ISDN-treated group (4). T, as baseline (before administration of kucat, ISDN or vehicle), m = 5 (each group). Blood samples were taken before treatment and 10, 30, 60, and 90 min after the treatment 21.74827.42%, p =0.512) (Table I, 2). After T, average NO level in kucai-treated anc ISDN-reated_ groups reduced down to certain level at Ty (Table 1, Fig. 2). 490 At To», compared fo control group, average levels of ‘NO was significantly higher (p = 0.018) in kucai-treated group, while that in ISDN-treated group was not significantly different (p= 0.436), Meanvshile compared to Jucartreated group, NO level in ISDN-treated group at ‘Ty were significantly lower (p = 0.028) (Table 1) Tt means that kucai-treated group serum keep remain NO levels compared to that of ISDN-treated group. Kucai showed longer effect up to 30 min, while ISDN showed shorter effect up to 90 min During the interval between TyeTy, average NO levels in ISDN-treated group decreased by 238.70165.72 uM, while in kucat treated group this figure was 221.264206.34 iM, Although the decrease in ISDN- treated group almost reach the initial level, comparison between the two groups didn't showed significant difference, p = 0.899 (Table 3). ‘The effet of kucai in increasing seram NO evel was. presumably dus to amide group in ts active compound, namely, tetramethyloxamide (Fidrianny et al, 2001). The mechanism of action was presumably resembles that of ISDN, CONCLUSION ‘The study results suggested that ethanol extract of ucai bulbs increased NO level significantly in experimental rats. Therefore, itis preassumed that the antihypertensive effect shown by kucai was, one of Which, due to its mechanism of action as vasodilator. The effect of kucai on the increased NO level was comparable to that of ISDN. ACKNOWLEDGMENTS ‘The authors wish to thank the Prodia Lab. for their helpful technical support. This study was supported by fa grant fiom ITB Research-Lembaga Penelitian and Pengabdian Masyarakat, Institut Teknologi Bandung Indonesia (No. 6004/KO1,3.2°PL2.1,577/2006),Int. REFERENCES: Balbatun, A., FR Louk and. Malinski, 2003. Dynamios| of nitric oxide release in the cardiovascular system, Acta Biochim Pol,, SO. 61-68, Balciogiu, A. C.J. Watkins and T.J. Maher, 1998. Use of ‘a hemoglobin-trapping approach in the determination of nitric oxide in im vitro and im vivo systems, Nourochom, Res, 23: 815-820. Baluchnejadmojarad, T., M. Roghani, H. Homayounfar and M. Hosseini, 2003, Beneficial effect of faquacous garlic extract on the vascular reactivity of streptozotecin-diabetic rats, J. Ethnopharmacol. 185; 139-144, Brovkowyeh, V., LW, Dobruski, S.Brokovyeh, 1 Dobrucki, CA, Do Nascimento, A. Burewiez and ‘T. Malinski, 1999, Nitric oxide release from normal and dysfunetional endothelium. J. Physiol. Pharmacol, 50 575-586, Fidianny, ,,K. Pacimawinata, S. Soetamo and E. Yulinal, 2001. Uji Efek Antihipertonsi Ekstrak Kucai (lfm schoenoprasum 1, Liliaceae), Prosiding Seminar ‘Nasional Tumbuhan Obat Indonesia XVIL 1st Edn,, Puslitbang Kimia Terapan IPI, ISBN: 979-8958-985 pp: 359-363, 491 Pharmacol, 4(6): 487-401, 2008 Furchgott, RF., 1983, Role of endothelium in responses of vaseular smooth musele. Cite. Ret, $35: Gharavi, N. and A.O. El-Kadi, 2003, Measurement of nitric ‘oxide in murine hepatoma hepalele? cells by reversed phase hple with Muorescence detection J, Pharma. Pharm, Sei. 6: 302-307, Katzung, B.O. andK. Chatterjee, 2007. Vasodilator and the ‘Treatment of Angina Pectoris. In: Basie and Clinical Pharmacology. 10h Edn, Katammg, B.G. (Bd), MeGraw Hill Publishing Co, Boston, pp: 183-187, ISBN: 978.007-126093-0, Klabunde, RE, 2004 Cardiovascular Physiology ‘Concepts. Ist Ean, Lippineatt Williams and Wilkins, Philadelphia, PP: 79-88, ISBN! 978-0781750301 Sumino, .,t. Nakamura, T. Kanda, K. Sato, T. Sakamaki, T. Takahashi, Y Saito, J. Hoshino, T. Kurashina and R. Nagai, 2000, Effect of enalapril on exhaled nite oxide in normotensive and hypertensive subjects Hypertension, 36; 934-940. Sun, J., X Zhang, M. Broderick and H, Fein, 2008, ‘Measurement of nitrite oxide production in biological system by using griess reaction assay. Sensors, 3: 276-284,