In the present study, BU was found to cause relaxation on the endothelium-intact aortic rings that had been precontracted with PE or KCl (Figure 1). This confirms the results reported in a previous study  that BU may reduce BP by vasodilation. BU was then further purified by column chromatography using solvent mixtures of different polarities. It was found that F1 demonstrated the highest vasodilatory activity and the effect was more profound (p < 0.05) when compared with BU-induced vasorelaxation. This indicates that most of the putative active compounds of BU appear to be concentrated in F1. Phenylephrine activates the α1-adrenoceptors in the VSM to cause the muscle contraction involving phospholipase C (PLC) and 1,2-diacylglycerol (DAG) [20, 21] while KCl causes the depolarisation of the VSM cell membrane, opening of VDCC and influx of Ca2+ in order to cause contraction . Both BU- and F1 induced relaxation in aortic rings precontracted with PE or KCl, suggesting that they might inhibit both the activation of α1-adrenoceptors and opening of the VDCC.
It is well known that vasoconstriction is initiated by elevated levels of the free cytoplasmic Ca2+ which can either be due to the influx of extracellular Ca2+ upon opening of the VDCC and ROCC located on the cell membrane [22, 23] or the release of Ca2+ from sarcoplasmic reticulum (SR) [24, 25]. To investigate whether F1 could interfere with the Ca2+ influx through VDCC, CaCl2-induced contraction in Ca2+-free with high K+ medium was used. Under high concentrations of K+, the added Ca2+ will cause the influx of Ca2+ through the VDCC . Whereas to investigate whether F1 could interfere with the Ca2+ through ROCC, Ca2+- and K+-free medium containing PE was used. Addition of PE in the Ca2+-and K+-free medium will cause the release of Ca2+ from intracellular store and the entry of Ca2+ into the cells through ROCC since VDCC are inactivated in the absence of K+[26, 27]. Results in Figures 3 and 4 show that preincubation with F1 significantly decreased the Emax of the CaCl2-induced contractions in both media, suggesting that the vasodilatory effect of F1 is caused by blocking the influx of Ca2+ via VDCC and ROCC.
Caffeine and NA were used to study the release of Ca2+ from intracellular sarcoreticular stores. Caffeine can stimulate the ryanodine receptors  and NA can trigger the activation of IP3 receptor located at the SR in order to cause the release of the Ca2+ from SR. In the present study, it is found that the vasoconstriction induced by both caffeine and NA were not affected by F1 (Figure 5). Therefore, the vasodilatory effect of F1 does not seem to be related to inhibition of ryanodine or IP3 receptors.
Potassium channels play a very crucial role in controlling the vascular contractility. Efflux of K+ due to the opening of the K+ channels in VSM will bring about membrane hyperpolarisation, which in turn, will lead to the closure of the VDCC and reduction of the Ca2+ entry, and finally cause vasodilation . In contrast, closure of the K+ channels will cause membrane depolarisation and vasoconstriction . Four distinct types of K+ channels have been identified in VSM, namely voltage-activated K+ channel, Ca2+-activated K+ channels, ATP-sensitive K+ channels, and inward rectifier K + channels) . As demonstrated by the results in Figure 6, F1 may be able to enhance or stimulate the opening of both voltage-activated and ATP-sensitive K+ channels, since preincubation with glibenclamide and 4-aminopyridine significantly attenuated its vasodilatory effect. On the other hand, Ca2+-activated K+ channel may not be involved in the vasodilatory effect of F1 since Rmax value of F1 was not significantly altered with the preincubation of TEA.
Nitric oxide is the primary EDRF formed in the endothelium that diffuses to the VSM to activate the soluble guanylyl cyclase. This enzyme will then catalyse the formation of cyclic guanosine monophosphate (cGMP), which in turn activates the protein kinase G (PKG). The PKG will cause the phophorylation of myosin light-chain kinase (MLCK) and subsequently decrease its activity, leading to the dephosphorylation of myosin light chain and thus causing vasorelaxation . In addition, NO also participates in the regulation of the level of smooth muscle free Ca2+, which is the primary determinant of contractions . Prostacyclin is the lipid compound derived enzymatically from fatty acid and it is the first EDRF that was discovered earlier than NO . It is synthesised in both VSM and endothelial cells . Results from the present studies show that removal of endothelium did not cause significant changes in the Rmax and pIC50 of F1-induced relaxation. This indicates that endothelium may not be involved in the vasodilatory effect of F1. Similarly, there were also no significant changes in the Rmax and pIC50 values for the aortic rings preincubated with L-NAME, ODQ, and methylene blue (Figure 7). These findings imply that F1 does not affect the endothelium-derived NO production as well as the NO-mediated pathway. To the contrary, significant reductions in both Rmax and pIC50 were observed in the aortic ring preincubated with indomethacin, the inhibitor of the cyclo-oxygenase that participates in the prostacyclin synthesis. Prostacyclin is synthesised in both endothelium and VSM. The vasodilatory effect of F1 was attenuated by the inhibition of cyclo-oxygenase but not by the removal of endothelium, suggesting that F1 may stimulate the release of prostacyclin from VSM.
The beta adrenoceptor acts by coupling with G protein. The activation of beta adrenoceptor stimulates adenylyl cyclase which functions as a catalyst for the conversion of adenosine triphosphate to cyclic adenosine monophosphate. This in turn leads to the activation of protein kinase A and vasodilation . Muscarinic receptors are stimulated by the postganglionic cholinergic neurons of either the parasympathetic or the sympathetic cholinergic systems that mediate smooth muscle relaxation, glandular secretion, and modulation of cardiac rate and force . Results from the present study show that there were no significant changes in the Rmax and pEC50 in the aortic rings preincubated with propanolol and atropine (Figure 8). This suggests that beta adrenoceptor and muscarinic receptor may not be involved in the vasodilatory effect of F1.
Phytochemcial screening of the compounds present in F1 detected high amount of flavonoids. Flavonoids are polyphenolic compounds that occur naturally in vegetables, fruits, seeds, nuts, barks and are an integral part of the human diet . Flavonoids belong to the low molecular weight group of polyphenol substances. The biochemical activities of flavonoids and their metabolites depend on their chemical structure and relative orientation of various moieties on the molecule. They can be grouped into 8 major classes by their chemical structures that include flavones, flavanones, flavonols, catechins, anthocyanidins, isoflavones, dihydroflavonols, and chalcones . Depending on their chemical structures, these flavonoids have been found to possess a wide range of pharmacological activities, such as antihypertensive [41, 42] and vasodilatory [43, 44] activities.
In the present study, although the phytochemical screening technique did not reveal the actual chemical structures of the active compounds in F1, it helped to show that the majority of compounds present in F1 are flavonoids. Based on the evidence that F1 exhibits vasodilatory activity and as well as containing flavonoids, it is logical to postulate that the vasodilatory effect of F1 could possibly be attributed to the presence of flavonoid. However more experiments need to be carried out in order to confirm this association and to identify the structure of the active compounds.