Abstract: The aim of this study was to determine the effect of ethanol extract of kucai bulbs on serum nitric oxide (NO) levels in rats, 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 kg-1 body weight (kucai-treated group); the second group with isosorbide dinitrate (ISDN) of 0.18 mg kg-1 body weight (ISDN-treated group) and the third group with vehicle only (control group). The results showed that at 30 min (T30) the mean NO level in kucai-treated group increased significantly (138.69 ± 169.38%) compared to baseline (T0); similarly, in ISDN-treated group, the mean NO level increased (212.08 ± 140.01%) significantly. At T90, average NO levels in kucai-treated group was significantly higher than those in control group (p = 0.018), while those in ISDN-treated group were not significantly higher than those in control group (p = 0.436). Compared to kucai-treated group, NO levels in ISDN-treated group at T90 was significantly different (p = 0.028). From this study it can be concluded that ethanol extract of kucai bulbs increased significantly serum NO level in experimental rats. Therefore, it is preassumed that antihypertensive effect shown by kucai is, one of which, due to mechanism of vasodilator action. The present results showed that kucai increased NO levels and this might be one mechanism of blood pressure lowering action of kucai.
INTRODUCTION
Nitric oxide (NO) is a chemical compound that plays a significant role in pathogenesis of several diseases, including hypertension. A physiological function of NO was discovered in the vasculature when it was shown that the endothelium-derived relaxing factor (EDRF) described by Furchgott and Zawadzki in 1980 is accounted for by formation of NO by endothelial cells (Furchgott, 1983; Sun et al., 2003). NO is produced by endothelium cell or nitrate group of drugs, 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 (iNOS) (Balbatun et al., 2003; Gharavi and El-Kadi, 2003). Free-radical NO produced will immediately react with molecular oxygen and water to form a variety of end-products, including nitrite (NO2–), nitrate (NO3–) and S-nitrosothiol (RSNO) (Gharavi and El-Kadi, 2003; Brovkovych et al., 1999). Free-radical NO is promptly inactivated by superoxide, Fe(II) and Fe(III), that are all in significant amount in biological system. The endothelium produced NO will diffuse into smooth muscle and activate guaianylate cyclase enzyme (atrial natriuretic peptide receptor A). This enzyme stimulates cyclic guanosine 3`,5`-monophosphate (cGMP) 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). Calcium ion release will further trigger 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 a medicinal plants that has been used traditionally in Indonesia for lowering blood pressure. Kucai possesses a good prospect to be developed as phytopharmaceutical product, so that it is expected as an alternative medication for hypertension. For this purpose, various steps of testing through both preclinical and clinical trials must be performed to study the efficacy and safety of kucai as antihypertensive agents. This study was conducted to determine the mechanism of kucai action on vascular relaxation (vasodilation) in experimental animals. There are 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 amount and its half-life (T1/2) is very short, i.e., 5 sec in biological fluid. Therefore, it is very difficult to measure NO products directly; instead, analysis of nitrate (NO3–) and nitrite (NO2–) are used to estimate NO level in biological fluid and cell culture medium due to their stability.
| Fig. 1: | Biosinthesize of nitric oxide (adapted from Cardiovascular Physiology Concepts, Klabunde, RE). NO is biosynthesized 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 cyclase enzyme, therefore stimulating cGMP 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. Calcium ion release will further make smooth muscle cell relaxation, reducing peripheral resistance, therefore vasodilation occurs |
Several methods have been developed in assessment of NO level by nitrite measurement; i.e., Griess colorimetric assay, chemiluminescence analysis, gas chromatograhic analysis, hemoglobin-trapping and fluorometric method. Apart from that, there are also ion-exchange, reverse-phase ion pairing, reverse-phase high-performance liquid chromatography method developed to measure nitrite and nitrate in biological system, using absorbance of UV-visible, conductivity, electrochemical or fluorescence (Gharavi and El-Kadi, 2003). In this study, determination of NO level was performed through measurement by Griess colorimetric assay. This method is considered adequately simple due to it is wide used to analyze some amount of biological samples, including plasma (serum), urine, cerebrospinal fluid, saliva and cell culture media (Sun et al., 2003). This study would determine NO level of rat serum as one of the methods to establish the mechanism of vasodilator action of kucai. In a earlier study on ethanol extract from kucai 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 study was performed in Pharmacology Laboratory-School 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 colorimetric Griess method, nitrite was first treated with diazotizing reagent e.g., sulfanilamide (SA) in acidic media to form a transient diazonium salt. This intermediate is then allowed to react with N-naphthylethylenediamine (NED) to form a stable azo compound. The intense purple color of the product allows nitrite assay and can be used to measured nitrite concentration as low as ~ 0.5 μM. The absorbance of this adduct at 540 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 Bandung, West Java, identified by botanists in the Herbarium Bandungense-School of Life Science and Technology, Institut Teknologi Bandung-Indonesia. The kucai extract was prepared with maseration-percolation method with 95% 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 g cadmium beads), ZnSO4 solution (2 mL of 30% (w/v)
ZnSO4), microcentrifuge tubes (50x1.5 mL microcentrifuge tubes),
Color Reagent No. 1 (7 mL sulfanilamide dissolved in 3 N HCl), Color Reagent
No. 2 (7 mL N-[1-naphtyl] ethylenediamine dihydrochloride dissolved in
deionized H2O), nitrite standard (1.5 mL 500 pmol μL-1
NaNO2, equivalent to 500 μM 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 M HCl and 125 mL of 0.1 M NH4OH).
Drugs: Isosorbide dinitrate (ISDN) tablets, produced by PT. Kimia Farma Tbk. (Bandung, Indonesia) was used in this study. ISDN was prepared as aqueous suspension and diluted with distilled water.
Animals: Fifteen male Wistar rats (weighing 250 to 300 g) were randomly selected and divided into three groups. The first group received a dose of 100 mg kg-1 of body weight of kucai, second group with a dose of 0.18 mg kg-1 body weight of isosorbide dinitrate (ISDN) and third group received only vehicle. Blood samples were taken before treatment and 10, 30, 60 and 90 min after the treatment (Sumino et al., 2000). This study was conducted in accordance with the Guideline for Care and Use of Animals Laboratory of School of Pharmacy Institut Teknologi Bandung.
Samples preparation: Blood sample was incubated at room temperature 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 μL of defrozen serum was adjusted to 190 μL with distilled water and then added with 10μL of 30% (w/v) ZnSO4 solution to make the total volume 200 μL. Final solution was then mixed by vortexing, incubated for 15 min at room temperature and centrifuged at 3,000-4,000 rpm for 5 min. The resulting supernatant was transferred to microcentrifuge tubes containing 0.5 g granulated 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 supernatant was tested for NO2 content.
Standard solutions preparation: Standard solution was prepared by diluting nitrite standard containing 1.5 mL 500 pmol μL-1 NaNO2 (equivalent to 500 μM NO). From the solution, serial nitrite standard solutions were prepared with NO concentration of 100, 50, 25, 10, 5, 1 and 0.5 μM (added with deionised water). Calibration curve thus made was used to determine nitrite levels.
Procedure: Cadmium beads were washed in their individual microtubes with H2O (2x1 mL); 0.1 M HCl (2x1 mL) and 0.1 M NH4OH, pH 9.6 (2x1 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 sample, 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 540 nm in microtiter plate reader. The standard curve was plotted and the sample concentration was estimated from the curve.
Statistical analysis: Statistical analysis was carried out by 2-way analysis of variance (ANOVA) by post-hoc test. Data are shown as mean values ± SD (standard deviation). A value of p<0.05 was considered statistically significant.
RESULTS AND DISCUSSION
Isosorbide dinitrate (ISDN) have been widely used as vasodilators to treat acute myocardial ischemia, their biological effects being due to the release of NO. ISDN have longer terminal elimination half life than other nitrovasodilator (20 min intravenously, 64 min sublingually) and has a high first-pass metabolism, broken down to 5-isosorbide-mononitrate and 2-isosorbide-mononitrate which are both more active than their parent compound. ISDN has been used as a drug standard in several studies on determination of nitric oxide in both in vivo and in vitro systems (Balciogiu et al., 1998; Baluchnejadmojarad et al., 2003).
The dose of extract used in this study was at dose of 100 mg kg-1 body weight. This dose was chosen on the basis of a earlier study suggesting that the kucai dose to have antihypertensive effect was 100 mg kg-1. While the dose of ISDN i.e., 0.18 mg kg-1 body weight was derived from conversion of human therapeutic dose.
The level of NO among the control, kucai-treated and ISDN-treated groups were not significantly different. Up to 10 min (T10) NO levels in kucai-treated, ISDN-treated and control groups were decreased compared to those of baseline (T0), but the decrease was not significant. After 10 min, NO levels increased in ISDN-treated and kucai-treated groups to their peaks (Table 1, Fig. 2).
At T30, mean NO levels of kucai-treated group increased significantly (% changes were 138.69 ± 160.38) compared to T0 (p = 0.044); similarly, that of ISDN-treated group increased significantly (% changes were 212.08 ± 140.01, p = 0.026), while in control group the increase was not significant (% changes were 21.74 ± 27.42%, p = 0.512) (Table 1, 2). After T30, average NO level in kucai-treated and ISDN-treated groups reduced down to certain level at T90 (Table 1, Fig. 2).
| Table 1: | Serum Nitric Oxide (NO) levels changes before and after administration of ethanol extract of Kucai |
| ISDN = Isosorbode dinitrate, a Compared to T0; b Compared to control group, a value of p<0.05 was considered statistically significant. At T30 NO level of kucai-treated group compared to ISDN-treated group p = 0.818 and at T90, p = 0.028 | |
| Table 2: | Percentage changes of serum NO levels at T30-T0 between different groups |
| ISDN = Isosorbide dinitrate, a Compared to control group, b Compared to ISDN-treated group, a value of p<0.05 was considered statistically significant. Changes were counted comparing to baseline (T0) serum NO level | |
| Table 3: | Comparison of serum NO levels changes between different groups |
| ISDN = Isosorbide dinitrate, a Compared to ISDN-treated group, bCompared to control group | |
| Fig. 2: | Effect of ethanol extract of kucai administration to NO serum level. Male Wistar rats divided randomly into three groups: control group (♦), kucai-treated group (■) and ISDN-treated group (▲). T0 as baseline (before administration of kucai, ISDN or vehicle); n = 5 (each group). Blood samples were taken before treatment and 10, 30, 60 and 90 min after the treatment |
At T90, compared to 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). Meanwhile compared to kucai-treated group, NO level in ISDN-treated group at T90 were significantly lower (p = 0.028) (Table 1). It 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 T30-T90, average NO levels in ISDN-treated group decreased by 238.70 ± 165.72 μM, while in kucai-treated group this figure was 221.26 ± 206.34 μM. 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 effect of kucai in increasing serum NO level was presumably due to amide group in its 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 kucai bulbs increased NO level significantly in experimental rats. Therefore, it is 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 a grant from ITB Research-Lembaga Penelitian and Pengabdian Masyarakat, Institut Teknologi Bandung Indonesia (No. 0004/K01.3.2/PL2.1.5/I/2006).