Endothelium-Independent Vasodilatory Effect of Sailuotong (SLT) on Rat Isolated Tail Artery

Background Sailuotong (SLT) is a standardized three-herb formulation consisting of extracts of Panax ginseng, Ginkgo biloba, and Crocus sativus for the treatment of vascular dementia (VaD). Although SLT has been shown to increase cerebral blood flow, the direct effects of SLT on vascular reactivity have not been explored. This study aims to examine the vasodilatory effects of SLT and the underlying mechanisms in rat isolated tail artery. Methods Male (250–300 g) Wistar Kyoto (WKY) rat tail artery was isolated for isometric tension measurement. The effects of SLT on the influx of calcium through the cell membrane calcium channels were determined in Ca2+-free solution experiments. Results SLT (0.1–5,000 μg/ml) caused a concentration-dependent relaxation in rat isolated tail artery precontracted by phenylephrine. In the contraction experiments, SLT (500, 1,000, and 5,000 μg/mL) significantly inhibited phenylephrine (0.001 to 10 μM)- and KCl (10–80 mM)-induced contraction, in a concentration-dependent manner. In Ca2+-free solution, SLT (500, 1,000, and 5,000 μg/mL) markedly suppressed Ca2+-induced (0.001–3 mM) vasoconstriction in a concentration-dependent manner in both phenylephrine (10 μM) or KCl (80 mM) stimulated tail arteries. L-type calcium channel blocker nifedipine (10 μM) inhibited PE-induced contraction. Furthermore, SLT significantly reduced phenylephrine-induced transient vasoconstriction in the rat isolated tail artery. Conclusion SLT induces relaxation of rat isolated tail artery through endothelium-independent mechanisms. The SLT-induced vasodilatation appeared to be jointly meditated by blockages of extracellular Ca2+ influx via receptor-gated and voltage-gated Ca2+ channels and inhibition of the release of Ca2+ from the sarcoplasmic reticulum.

contributor to stroke and dementia in aged mice [7]. erefore, interventions that can induce vasodilation or suppress vasoconstriction could be useful in reducing the progression of CVD. Sailuotong (SLT) is a standardized three-herb formula Chinese herbal medicine designed for the management of VaD associated with ischemic stroke [8,9]. e SLT formula consists of standard extracts of specific dosages of Panax ginseng C. A. Meyer (ginseng), Gingko biloba L. (ginkgo), and Crocus sativus L. (saffron). e chemical profile and ratio of the three herbal extracts had been determined and studied in detail previously [10,11]. e cognitive enhancing effect of SLT has been demonstrated in both preclinical and clinical studies. In a chronic cerebral hypoperfusion model induced by bilateral common carotid artery ligation in rats, eight-week treatment of SLT significantly suppressed hypoperfusion-induced cognitive impairments, and this change is associated with reduction of activity of cholinesterase and increased acetylcholine (ACh) levels and superoxide dismutase (SOD) activity the brain tissue [8]. Our previous work also showed that SLT protected cultured human vascular endothelial cells against hydrogen peroxideinduce injury via increase in SOD activity [12]. e potential beneficial effects of SLT for VaD were demonstrated in two separate phase II randomized, double-blinded, placebocontrolled clinical studies. In these studies, treatment with SLT was shown to significantly improve cognitive function and cerebral blood flow to the inferior frontal and anterior temporal lobes in patients with VaD [11,13].
Reports from animal and clinical studies have indicated that the clinically beneficial effect of SLT is, at least, partially associated with an increase in cerebral blood flow [14,15]. However, the underlying mechanism(s) associated with these SLT-mediated cerebral blood flow increase remain to be determined. In this study, we hypothesised that SLT has direct modulatory effects in vascular reactivity. erefore, this study was aimed to evaluate the vascular effects and the underlying mechanisms of SLT using rat isolated tail artery.

High-Performance in-Layer Chromatography (HPLC)
Analysis of the SLT Formula. HPLC-PDA was employed to profile the phytochemical composition of the SLT extract used in the study. e HPLC-PDA analysis was performed on a Prominence-I LC-2030 3D Plus Shimadzu HPLC system controlled by Lab Solutions software (Shimadzu, Australia). Separation was achieved using a Shimadzu Shim-pack GIST (Shimadzu, Australia) reverse phase C18 column (4.6 × 150 mm I.D., 5 μm) maintained at 40°C. e SLT extract was dissolved by sonication in 30% aqueous acetonitrile for 30 min at 5 mg/ml. Individual solutions of standards, crocin, ginsenoside Re, ginsenoside Rg1, ginsenoside Rd, quercetin, kaempferol, and isorhamnetin were prepared to 1 mg/10 ml in 30% aqueous acetonitrile for identification and combined for analysis. e sample and mixed standard were syringe-filtered with 0.45 μm PTFE. e SLT HPLC-PDA profiles were generated by 20 μl injection. e mobile phase consisted of 0.1% (v/v) aqueous formic acid (mobile phase A) and 0.1% (v/v) formic acid in acetonitrile (mobile phase B). e gradient program was 10% B for 1 min with a linear increase, to 45% B at 45 min, and then, a wash and re-equilibration. e mobile phase flow rate was maintained at 1.1 ml/min. e PDA was set to acquire absorbance data from 190 to 800 nm.

Animals.
A total of forty 12-week-old, male Wistar Kyoto (WKY) rats weighting 200-250 g were obtained from the Animal Resource Centre (Canning Vale, Western Australia). All experimental animals were housed under a 12:12 hour light-dark cycle (relative humidity: 50-60%; temperature: 22 ± 1°C) and were given standard chow and water ad libitum throughout the experimental period. On the day of experiment, the rats were sacrificed by CO 2 asphyxiation, and vascular preparations were isolated under a dissection microscope. All animal protocols conformed to the Guide for the Care and Use of Laboratory Animals by the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose [16]. Institutional ethics approval (Approval number: A12041) was obtained from Western Sydney University prior to commencement of the study.

Isometric Tension Measurement.
Isometric force measurements and experimental protocols were performed on tail artery isolated from the WKY rats as described previously with slight modifications [5,17]. e isometric force of the arterial ring segments was measured using a multiwire myograph system (Model 620M; Danish Myo Technology, DMT, Denmark). During the isometric tension measurement, the chambers of the myograph were filled with Krebs-Henseleit solution (mmol/L: NaCl 118.0, KCl 4.7, K 3 PO 4 1.2, MgSO 4 1.2, NaHCO 3 25.0, CaCl 2 1.8, glucose 11.0), unless stated otherwise. e Krebs-Henseleit solution were prepared and used as follows: All solutions were warmed to 37°C and bubbled with carbogen (95% O 2 and 5% CO 2 ) before they were added to the myograph chamber. Each chamber containing 5 ml of Krebs buffered solution was bubbled with 95% O 2 and 5% CO 2 and maintained at a pH of 7.4 at 37°C throughout the experiment. Arterial rings (1.5-2.0 mm length) were mounted in the organ chambers, and normalized passive resting force and the corresponding diameter were determined for each preparation from its own length-pressure curve and stretched to their optimal lumen diameter (i.e.,∼90% of the diameter which is equivalent to a transmural pressure of 100 mm Hg). e rings were, then, left for a stabilization period of about 30 minutes until a stable base line tone was obtained before beginning of the experiment. e presence of the endothelium was tested with acetylcholine (1 μM) after preconstriction with phenylephrine (PE) (1 μM). Tissues which showed a relaxation to acetylcholine of less than 60% of the phenylephrine contraction were not considered with intact endothelium and, therefore, not employed in this study. Responses were recorded using computerized data acquisition and recording software. e acute and direct vasodilatory effect of SLT was evaluated on rat isolated tail artery. PE (1 μM) was applied to induce a steady contraction. When the contraction reached a plateau, the SLT extract (0.1-5000 μg/ ml) was added cumulatively to the isolated tail artery rings with or without the presence of L-NAME (20 μM) to obtain a concentration-response curve.

Experimental Protocols of Vascular Tension
e relaxation effect was calculated as the percentage of the contraction in response to PE.

Protocol 3: Calcium-Induced Vasoconstriction in Rat Isolated Tail Arteries.
e effect of SLT on calcium influx induced by two different agents was evaluated. e preparations were initially contracted with either KCl (80 mM) or PE (10 μM) to determine maximum contraction of each preparation in normal Krebs solution. e preparations were washed with a calcium-free solution (identical concentration of EGTA substituted for Ca 2+ in a normal Krebs solution) until fully relaxed. To exhaust the intracellular calcium storage, the preparations were stimulated with either PE (10 μM) or KCl (80 mM) repeatedly, with calciumfree solution wash in between, until any contractile response disappeared. e preparations were, then, incubated with SLT at various concentrations (500, 1,000, and 5,000 μg/ml) for 30 min. After incubation, a single dose of the contractile agent (either KCl (80 mM) or PE (10 μM) was added to the chamber, and calcium (0.001-3 mM)-induced contraction concentration response curves were constructed.

Protocol 4: Phenylephrine-and Potassium Chloride-(KCl-) Induced Vasoconstriction in Rat Isolated Tail Arteries.
In order to assess the effects of SLT on the contraction induced by phenylephrine and potassium chloride (KCl), concentration-response curves of PE and KCl were constructed with or without the presence of SLT at various dosages (500, 1,000, and 5,000 μg/ml). e arterial preparations were allowed to preincubate with SLT (0, 500, 1,000, and 5,000 μg/ml) for 30 minutes' prior the construction of the PE or KCl-induced vasoconstriction concentration-response curve.

Protocol 5: Role of Intracellular Calcium-Induced Vasoconstriction in Rat Isolated Tail Arteries.
To clarify the role of intracellular Ca 2+ release from the sarcoplasmic reticulum (SR) in the relaxant effect of SLT, the isolated tail artery rings were exposed to a Ca 2+ -free solution for 15 minutes before the application of 1 μM of PE to induce the first transient contraction (Con 1). e rings were, then, washed with a normal Krebs solution for three times and incubated in the normal Krebs solution for 45 minutes to allow refill of the intracellular Ca 2+ storage. After the 45-minute incubation, the medium was rapidly replaced with a Ca 2+ -free solution and allowed to incubate for 15 minutes. e second contraction (Con 2) was induced by 1 μM of PE with or with the presence of SLT (500, 1,000, or 5,000 μg/ml). e ratio of Con 2 to Con 1 was calculated.

Statistical
Analysis. Data are expressed as means ± S.E.M., and n denotes the number of replications for each data point. Relaxation was expressed as the percentage of the contraction elicited by phenylephrine or KCl. Statistical comparisons were performed using the t-test or two-way analysis of variance (ANOVA), where appropriate. Differences were considered to be statistically significant at P < 0.05. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., USA).

High-Performance in-Layer Chromatography (HPLC) Analysis of the SLT Extract.
e active ingredients and contents in SLT have been reported in details previously [10,11]. Six major bioactive compounds were analyzed using HPLC. Similar to these studies, our results showed that all these major bioactive compounds, including ginsenosides Rg1, ginsenosides Re, ginsenosides Rb1, quercetin, isorhamnetin, and crocin, are present in the SLT extract ( Figure 1). e chromatogram is visualized at different wavelengths corresponding to the optimal wavelength to detect the relevant group of markers. SLT is reported to contain ginsengs as ginsenosides Rg1 (2.4 mg/capsule), ginsenosides Re (1.6 mg/capsule), and ginsenosides Rb1 (4.49 mg/capsule). e ginsenosides were confirmed to be present in similar concentrations by matching retention time and the UV spectrum to the reference standards as shown in Figure 1 at 203 nm. e peak size is not reflective of abundance as the different ginsenosides have unique molar absorptivity. SLT is reported to contain Ginkgo biloba, with Evidence-Based Complementary and Alternative Medicine markers quercetin (2.71 mg/capsule) and isorhamnetin (1.55 mg/capsule). ese were identified in the SLT extract used in this study as shown in Figure 1 at 370 nm. SLT is reported to contain saffron, with the marker crocin (1.7 mg/ capsule). Crocin was identified in the SLT extract used in this study as shown in Figure 1 at 440 nm. e HPLC analysis of the SLT extract supplied by Shineway was consistent with other reports [10,11].

Effects of Potassium Channel Blockers on SLT-Induced
Vasodilatation in Rat Isolated Tail Arteries. Opening of potassium channels plays an important role in vasodilatation. We examined the involvement of potassium channels on the SLT-induced relaxation in the rat isolated tail arteries. Tetraethylammonium (TEA, a nonselective potassium channel blocker) (1 mM), glibenclamide (Glib, an ATPsensitive potassium channel blocker) (3 μM), and clotrimazole (K Ca , a calcium-activated potassium channel blocker) (5 μM) were tested. As shown in Figure 2, preincubation of potassium channel blockers did not  Evidence-Based Complementary and Alternative Medicine significantly alter the SLT-induced vasodilatation in PE (1 μM)-preconstricted rat isolated tail artery (n � 5-6) ( Figure 3).

Effects of the SLT Extract on Calcium-Induced Vasoconstriction in Rat Isolated Tail Arteries Stimulated by
Phenylephrine or KCl. e effect of the SLT extract on calcium-influx-induced vasoconstriction on rat isolated tail arteries was assessed with two vasocontractile stimulants (phenylephrine and KCl) by reintroducing calcium (0.001-3 mM) into the calcium-free buffer. In the phenylephrine (10 μM)-stimulated tail arteries, cumulative administration of calcium chloride (0.001-3 mM) induced a vasoconstriction in a concentration-dependent manner, with 29.167 ± 7.23% contraction observed at 3 mM (n � 5). Preincubation of the SLT extract (500, 1,000, and 5,000 μg/ ml) dose-dependently suppressed the calcium-induced vasoconstriction in the phenylephrine (10 μM)-isolated tail arteries. SLT at 5,000 μg/ml and nifedipine (10 μM) markedly suppressed the calcium-induced vasoconstriction (Figure 4(a)).

Discussion
Vascular dementia (VaD) is considered to be caused by chronic cerebral ischemia which induces neuronal damage, resulting in a decline in cognitive function eventually. While the development and progression of cognitive impairment and VaD are certainly multifactorial, it has been suggested that cerebrovascular dysfunction leads to reduction of cerebral blood flow which is closely associated with cognitive impairment in diabetes [18,19]. Sailuotong (SLT) is a standardized three-herb formula designed for the management of VaD. Although data from animal and clinical studies have indicated that the clinically beneficial effect of SLT is closely associated with an increase in cerebral blood flow [11][12][13], the acute and direct modulatory effects and the underlying mechanisms of actions of SLT in vascular reactivity have not been studied. Results from the current work provide evidence that the SLT extract induces vascular relaxation in rat isolated tail arteries. Furthermore, our results showed that this SLT-induced vasodilatation is mediated via -10 * * * * * * * * * * * * * * * * * * * * * * *  Evidence-Based Complementary and Alternative Medicine endothelium-independent mechanisms including inhibition of extracellular Ca 2+ influx and release of Ca 2+ storage from the sarcoplasmic reticulum. e individual herb extracts of SLT have been shown to induce vasodilatation via endothelium-mediated mechanisms [20]. For example, crocetin, a carotenoid derived from saffron, has been shown to exert vasomodulatory effects via an endothelium-dependent mechanism [21]. Similarly, previous studies have demonstrated that ginseng produces its vasodilatory effect through eNOS activation in the endothelium [22,23]. In contrast, our results revealed that inhibition of the endothelium by L-NAME failed to suppress the SLT-induced vasodilatation, indicating that the SLTinduced relaxation is largely mediated by an endotheliumindependent mechanism.
is relaxation was most likely caused by the complex constituents within the SLT extract which acted directly on the vascular smooth muscle cell (VSMC). For instance, the Ginkgo biloba extract (GBE) has been shown to induce vasodilation via multiple mechanisms, including inhibition of calcium influx through the L-type calcium channel and the activation of NO release in the rat isolated aorta [24]. Indeed, it has been shown that, while all the Ginkgo extract constituents, including bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, quercetin, and rutin, have vasodilating action, each of them can contribute to GBE-induced vasorelaxation with different mechanisms and signaling pathways [25].
Since our results indicate that the SLT-induced relaxation was not mediated via the endothelium, other endothelium-independent mechanisms were considered. Vascular tone and reactivity regulation are primarily controlled by the changes of intracellular calcium within the vascular smooth muscle cell (VSMC) [26]. A number of calcium channels and mechanisms are responsible for the increase of the intracellular calcium level in VSMC [27][28][29][30], and many herbs and their isolated compounds have been shown to induce vasodilatation via inhibition of calcium influx in the VSMC [24]. For instance, the Ginkgo biloba extract has been shown to induce aortic relaxation via inhibition of calcium influx through the calcium channel [24]. Similarly, ginsenoside Rg (3), an active ingredient of Panax ginseng, has been shown to inhibit the L-type calcium channel in Xenopus oocytes in a dose-and voltage-dependent manner [31]. In the current study, reintroduction of calcium to phenylephrine-or KCl-stimulated isolated tail artery bathed in a calcium-free solution caused contractions in a concentration-dependent manner. ese results clearly demonstrated the role of calcium influx in vasoconstriction in response to both receptor-mediated (i.e., phenylephrine) and voltage-mediated (KCl) stimulations. SLT dose-dependently inhibited the calcium-influx-induced vessel contraction to both phenylephrine-and KCl-stimulated tail arteries, suggesting SLT was likely to inhibit both receptorand voltage-mediated calcium influx. In line with this, the ability of SLT to suppress both phenylephrine-and KClmediated responses were clearly demonstrated in the phenylephrine-and KCl-induced vasoconstriction experiments, where SLT markedly suppressed the vasoconstriction caused by both agents. erefore, our results suggested that SLT suppressed calcium influx by inhibition of both the voltage-operated calcium channel (VOCC) and receptoroperated calcium channel (ROCC).
Although the effects of SLT on intracellular calcium storage regulation in VSMC have not been reported previously, the individual components of SLT have been shown to suppress increase of the intracellular calcium level via inhibition of both influxes of extracellular Ca 2+ and release of intracellular Ca 2+ storage. In our study, SLT inhibited the PE-induced transient contraction in the isolated tail artery, indicating that SLT suppressed the vasoconstriction via inhibition of the release of Ca 2+ from the sarcoplasmic reticulum. Similar to our study, ginsenoside-Rd, a main active constituent of Panax ginseng, has been shown to suppress phenylephrine-induced contraction in rat isolated aorta via inhibition of both receptor-and store-operated calcium channels in VSMC [22]. Yang et al. [32] have shown that the antiplatelet and antithrombosis properties of saffron were mediated by a mechanism involving inhibition of calcium mobilization via reducing both intracellular calcium release and extracellular calcium influx [32].
Many herbal medicines have been shown to produce their cardiovascular protective effects via the opening of potassium channels in VSMCs, such as the ATP-sensitive potassium channel (K ATP ) and calcium-activated potassium channel (K Ca ) [33,34]. For example, Li et al. [35] showed that ginsenosides (GS), an extract of Panax ginseng, induced aortic relaxation and is, at least, partly mediated by the opening of the K Ca channel in the VSMC [35]. Similarly, activation of the potassium channel has also been demonstrated in ginsenoside Rg3-mediated endothelium-dependent relaxation in rat isolated aorta [36]. e Ginkgo extract has been shown to relax smooth muscle cell via a TEAsensitive (a nonselective potassium channel blocker) mechanism [37] and opening of the K Ca channel [38], while other studies suggested that Ginkgo induces vasodilatation via inhibition of calcium influx [24,39] and activation of eNOS [40]. In this study, preincubation of TEA (a nonselective potassium channel blocker), glibenclamide (an ATPsensitive potassium channel blocker), and clotrimazole (a calcium-activated potassium channel blocker) did not have any effects on the SLT-induced relaxation in the rat isolated tail artery, suggesting that the relaxation was independent of potassium channels opening. Although the effects of the individual components of SLT on the potassium channel have been reported previously [36], it is not uncommon that Chinese herbal medicine can induce relaxation independent of potassium channel activation. For example, the Danshen aqueous extract and its active component, Salvianolic acid B, have been shown to produce their vasorelaxant effect independent from potassium channel opening [41].
ere are three limitations in the current study. First of all, although numerous studies have demonstrated that the reactivity profile of the rat tail artery resembles the human peripheral arteries [42][43][44] and have been used as an ex vivo model of resistance artery which can be extrapolated to human peripheral vascular disease [45], quantitative measurement of cerebrovascular reactivity should be employed in the future study. In addition, while our current work showed that acute SLT administration did not induce endothelium-dependent vasodilatation in the vessels isolated from a healthy animal, previous work from our group has demonstrated that SLT protected the cultured human endothelial cell from oxidative injury [12], indicating that the endothelium modulatory effects of SLT can only be observed under diseased conditions. Hence, endothelial effects of SLT in disease models should be studied in the future. Finally, while the active components of SLT have been shown to be absorbed into systematic circulation [46], any effects mediated by the metabolites of SLT may be overlooked by the ex vivo model used in the current study. A detailed metabolomics study is required to identify and explore the possible contribution of metabolites of SLT on vasculature.

Conclusions
In conclusion, we demonstrated that acute administration of the SLT extract induced vasodilatation in rat isolated tail artery. SLT-induced vasodilatation appeared to be principally mediated via endothelium-independent mechanisms, including blockage of extracellular Ca 2+ influx and inhibition of release of Ca 2+ from the sarcoplasmic reticulum. Results from this work provide direct evidence to explain the beneficial effects of SLT observed in clinical trials. Data Availability e data that support the findings of this study are available from the corresponding author (SWS) upon reasonable request.

Ethical Approval
All animal protocols conformed to the Guide for the Care and Use of Laboratory Animals by the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose. Institutional ethics approval (Approval number: A12041) was obtained from Western Sydney University prior to commencement of the study.

Conflicts of Interest
As a medical research institute, NICM Health Research Institute receives research grants and donations from foundations, universities, government agencies, individuals, and industry. Sponsors and donors also provide untied funding for work to advance the vision and mission of the institute. e authors declare no conflicts of interest.