In the present study, the extracts (leaves, stems, and roots) from C. citratus
were prepared as methanolic extracts. Despite the traditional practice of using aqueous extracts, methanol was chosen as the solvent so as not to miss any possible active compounds since it will dissolve both polar and some nonpolar constituents [20
]. In addition, methanol is a common water-miscible solvent, and the solubility percentage in water is 100% with polarity index of 5.1 [21
]. It needs to be pointed out that the methanol was evaporated and the dried extract residue was subsequently reconstituted in distilled H2
O for all studies using the aortic ring preparation.
This paper reports the vasorelaxant properties of citral and methanolic extracts of leaf (LE), stem (SE), and root (RE) of C. citratus
using isolated rat aortic rings and the possible mechanism(s) involved. The present observation of the vasorelaxant effect of citral on the PE-induced contraction in SHR aortic rings was in agreement with earlier studies that reported vasodilatory and antihypertensive effects of terpenoids [22
]. A similar effect was shown by the leaf and root extracts suggesting that citral may be the main constituent in these extracts. The current study, however, indicates the presence of other compounds in both the leaf and root extracts since the vasorelaxant effect was observed in the aortic rings of both SHR and WKY rats, whereas citral showed the effect only in SHR.
Citral, LE, and RE significantly attenuated the contractile response induced by the selective α1
-adrenoceptor agonist, PE, suggesting that these test materials may modulate the endothelial-derived relaxing factors (EDRFs) that include endothelial-derived NO (EDNO) and prostacyclin (PGI2
]. It is well established that NO is a major EDRF that plays a central role in the maintenance of vascular tone [24
]. When released from the endothelium, NO diffuses into the smooth muscle and then triggers the formation of cyclic guanosine monophosphate (cGMP), increasing the cGMP levels that lead to vasorelaxation. A reduced NO production by vascular endothelial cell is closely associated with endothelial disease or injury that has been proposed to be an important causative factor in cardiovascular diseases, especially in the development of arteriosclerosis and hypertension [26
The involvement of NO in the relaxant effect produced by citral, LE, and RE in isolated aortic rings was investigated by incubating the E+ rings with the NO synthase inhibitor, L-NAME, prior to inducing contraction with PE and the subsequent addition of the test materials. The citral-induced smooth muscle relaxation on the PE-contracted rings was observed to be significantly reduced in the presence of L-NAME. These data provided evidence that NO may be involved in the vasorelaxant effect of citral. It also supports the hypothesis that citral may stimulate endothelial NO production and/or release. Whether citral is directly interacting with endothelial NO synthase or with other factors, which may increase the endothelial NO synthase activity, remains to be further investigated. Under the same condition, the relaxation caused by LE and RE was not attenuated. These results suggest that NO, a modulator of the vascular function [27
], may partially play a role in the vasodilator effect of citral but not that of LE and RE. In fact, RE caused a significantly greater relaxant effect in the presence of L-NAME. The reason for this change remains to be elucidated.
Besides NO, prostaglandin (PG) is also produced by endothelial cells to counteract the vasoconstriction induced by the sympathetic nerve endings and humoral vasoconstrictors. After the discovery of EDRF, it was demonstrated that endothelial cells also can generate cyclooxygenase- (COX-) derived vasoconstrictor substances in canine veins and in arteries of SHR [28
]. Thus, by secreting relaxing and constricting factors, the endothelium can induce dilation or constriction in response to sheer stress and a variety of endogenous vasoactive substances that are either produced systemically or generated locally by vascular tissues or circulating in the blood.
), a major vasodilatory COX product, is produced by the intimal layers of the vascular wall [27
]. In the present study, the involvement of PGI2
in the relaxant effect of the test materials on intact aortic ring preparations was investigated by preincubating the rings with indomethacin. Indomethacin is an inhibitor of COX and will inhibit the synthesis of various PGs including PGI2
and markedly inhibits the transient relaxation induced by arachidonic acid [30
]. Indomethacin did not seem to affect the relaxant effect of citral. On the other hand, the relaxant effect produced by RE was significantly increased, thus, suggesting that PGI2
did not play an important role in the vasorelaxant response induced by RE. We cannot offer a good explanation for the increased vasorelaxant effect in this paper. Perhaps RE contains a non-PG vasodilator and a small amount of vasoconstricting PG such as thromboxane A2
such that in the presence of indomethacin the opposing vasoconstrictor response was removed giving rise to a net increase in vasorelaxant response.
Interestingly, the presence of indomethacin caused a reversal of the relaxant effect of LE, causing instead a contraction of the aortic rings. It could be postulated that the LE may have a vasoconstrictor as well as vasorelaxant agents as its constituents and that the relaxant effect is more dominant. The relaxant effect may partly be due to PGI2 or other PGs, and when the synthesis of PGs was inhibited by indomethacin, the non-PG vasoconstrictor effect became unmasked giving rise to contraction.
As with other muscles, the smooth muscle requires Ca2+
to contract. It is well established that the influx of external Ca2+
through specific Ca2+
channels or Ca2+
release from internal stores plays an important role in excitation-contraction coupling of smooth muscles. By binding to specific membrane receptors, PE induces Ca2+
influx through receptor-operated channels causing tonic contraction [31
] and stimulates the formation of inositol triphosphate (IP3
) that binds to and opens specific IP3
-receptor channels in the sarcoplasmic reticulum membrane, inducing Ca2+
release from intracellular storage sites and causing phasic contraction [32
When the aortic rings were incubated in a Ca2+-free Krebs solution and PE was added into the bath there was a transient increase in contraction due to release of Ca2+ from intracellular storage sites. The addition of increasing doses of CaCl2 solution into the organ bath caused a dose-dependent increase in contraction of both the E+ and E− aortic rings. However, when the rings were Pretreated with citral, LE, and RE, both the transient PE-induced contraction and the CaCl2-induced contraction were abrogated. Based on these findings, it may be postulated that citral, LE, and RE can either block the entry of Ca2+ from the extracellular space possibly via receptor-operated calcium channel or the Ca2+ release from intracellular storage sites. Further studies are required to understand the exact mechanism by which citral and the extracts affect the intracellular Ca2+ levels.