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Cerebral vasodilator properties of Danshen and Gegen: a study of their combined efficacy and mechanisms of actions.

ABSTRACT

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (Cl) of 1.041 at [ED.sub.50], which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a [K.sub.ATP] channel inhibitor glibenclamide, a non-selective [K.sup.+] channel inhibitor tetraethylammonium (TEA), and by a combination of [K.sup.+] channel inhibitors (glibenclamide + TEA + iberiotoxin + 4-aminopyridine + barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of KATP currents and inhibited [Ca.sup.2+] influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited Ca[Cl.sub.2]-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of [K.sub.ATP] channels and inhibition of [Ca.sup.2+] influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.

Keywords:

Danshen

Gegen

Vasodilatation

Basilar artery

Stroke

Combination index

Introduction

Danshen is the dried root of Salvia miltiorrhiza. This herb has properties of promoting blood circulation, relieving blood stasis, clearing heat from the blood, resolving swelling and tranquillizing the mind. It is often used in decoction preparations to treat dementia and cardiovascular diseases such as angina pectoris, myocardial infarction, and hyperlipidemia (Ji et al. 2000; Cui and Li 2003; Cheng 2006). Gegen is the dried root of Pueraria lobata. This herb has been ascribed with a plethora of biological activities including anti-pathogenic, anti-hypertensive, anti-platelet, anti-inflammation, anti-apoptotic, and anti-diabetic activities (Song et al. 2007; Wong et al. 2011). It is often used for improving liver function, enhancing alcohol detoxification processes, regulating cardiac functions, and aiding weight loss (Wong et al. 2011).

The combined use of drugs for treatment of diseases is gaining popularity in western medicine with the belief that it could provide better efficacy over monotherapy. In recent years, there is remarkable progress in the treatment of cancer and infectious diseases based on such approach (Risberg et al. 2011). Practitioners of TCM have utilized herbal combinations for treatment of diseases for thousand of years. One such example is TCM formulations that contained Danshen and Gegen (DG), which are often used for treatment of coronary heart diseases (Zhang et al. 1991; Chen 2002; Ren and Luo 2006). DG decoctions have been shown to possess anti-oxidative and vasodilator properties (Lam et al. 2005), modulate key early events in atherosclerosis (Sieveking et al. 2005), and improve arthrogenic pathophysiology (Chan et al. 2006; Cheung et al. 2012). Furthermore, a double-blind clinical trial showed that a DG decoction improved lipid profile, vascular function and structure in coronary patients (Tam et al. 2009). These cardiovascular benefits of DG formulations are likely to be applicable to the cerebrovascular system, but hitherto, there is limited investigation and evidence supporting their use in cerebrovascular disorders.

Ischaemic stroke is a leading public health problem, which is second to heart disease as a worldwide cause of death. The cerebral basilar artery is one of the arteries that supplies the brain with oxygen-rich blood. Ischaemic stroke due to obstruction of the basilar artery carries a poor prognosis as it causes hypoxia and ischaemia of the brainstem that could lead to paralysis of all extremities, heavy disturbances in sensation, difficulty in swallowing and difficulty in respiration. We have previously shown that a DG decoction possesses dilator properties on the rat basilar artery (Lam et al. 2010), indicating that it could potentially be useful in treatment of ischaemic stroke. A recent report also suggests that, depending on the parameters being measured, the combined use of Danshen and Gegen could result in synergistic, additive, or even antagonistic efficacies (Cheung et al. 2012). However, which of these outcomes are true for the cerebral vasodilator effects of our DG decoction is unknown. Therefore, in the present study, we have compared the efficacies of this DG decoction with its parent herbs, Danshen and Gegen, in relaxing the rat isolated basilar arteries. Furthermore, we have performed pharmacological, electrophysiological, and confocal microscopic studies to elucidate their mechanisms of actions.

Materials and methods

Chemicals

The raw herbs of Danshen and Gegen were purchased from local herbal shops in Sichuan and Guangdong province, respectively. Iberiotoxin was purchased from Tocris (UK). All other drugs were purchased from Sigma, USA. Glibenclamide, nifedipine and ODQ were dissolved in DMSO. All other drugs were dissolved in distilled water and stock solutions were stored at -20[degrees]C.

Preparation of extracts

Water extracts of Danshen, Gegen, and DG (ratio 7:3) were prepared as previously described (Cheung et al. 2012). Identification and quantification of the major components in the DG extract including danshensu (DSS), salvianolic acid B (SAB), protocatechuic aldehyde (PCA), puerarin, daidzin, and daidzein were performed by a RP HPLC-PAD method according to our previous study (Chiu et al. 2011)

Vasorelaxant effects on rat basilar artery

Rat basilar arteries were prepared for pharmacological studies on tension changes as previously described (Deng et al. 2012). The combination effects of Danshen and Gegen were analyzed by Calcusyn software to produce combination indexes (C/s) at varying effective doses (ED) (Chou 2006). A Cl value between 0.9 and 0.85 would suggest a moderate synergy, whereas that in the range of 0.7-0.3 is indicative of a clear synergistic interaction between the drugs. On the other hand, a Cl value in the range of 0.9-1.1 would suggest an additive effect, and a Cl value exceeding 1.1 would indicate an antagonistic effect.

Effects on [K.sup.+] current in rat cerebral vascular smooth muscle cells

Single basilar artery smooth muscle cells were prepared as described by Wu et al. (2005), and [K.sub.ATP] current amplitudes were recorded as described by Lam et al. (2006a).

Effects on intracellular calcium [[Ca.sup.2+].sub.i] levels in rat cerebral vascular smooth muscle cells

Single basilar artery smooth muscle cells were prepared as described by Wu et al. (2005). A real-time confocal microscope (Olympus FV 1000) was used to detect changes in intracellular calcium [[Ca.sup.2+].sub.i] levels. The myocytes were loaded with 5 [micro]M of fluo4/AM diluted in Krebs solution for 0.5 h at 37[degrees]C. They were then perfused for 10 min with [Ca.sup.2+]-free Krebs solution containing 1 mM EGTA to chelate residual [Ca.sup.2+], 10[micro]M cydopiazonic acid (CPA) to deplete SR [Ca.sup.2+] stores, and with one of the following drugs: Danshen, Gegen, DG or nifedipine. Influx of Ca2+ was measured before and after restoration of extracellular [Ca.sup.2+] to 2.5 mM. Cell images were captured continuously every 4 s, and changes in [[Ca.sup.2+].sub.i] were determined via ratiometry.

Statistical analysis

All data are presented as means [+ or -] SEM vasodilator responses were measured as percentage reduction on the U46619-precontracted tone and CaCl2-induced vasoconstrictions were measured as tension changes in grams. GraphPad Prism software was used to fit sigmoidal curves and to determine values of [IC.sub.50] and maximum inhibition of the precontracted tone (Imax). [[Ca.sub.2+].sub.i] was expressed as the ratio of fluorescent intensity. The n value refers to the number of basilar arterial ring preparations or single vascular smooth muscle cells used. Statistical comparisons were performed using one-way or two-way ANOVA, where appropriate, and differences between [IC.sub.50] and Imax values were compared by Bonferroni post hoc test. Differences between area under the curves (AUC) for [[Ca.sup.2+].sub.i] fluorescent intensity were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. Differences were considered to be statistically significant at a value of p < 0.05.

Results and discussion

The development of cerebral vasospasm remains the most common and troublesome complication of aneurysmal subarachnoid haemorrhage. The exact pathophysiological mechanism responsible for the vasospasm remains uncertain, but it begins most often on the third day after the ictal event and reaches the maximum on the 5th to 7th postictal days. Several therapeutic modalities have been employed for preventing or reversing cerebral vasospasm. These include the hypervolaemic-hypertensive-haemodilution (Triple H) therapy, balloon and chemical angioplasty with intra-arterial injection of vasodilators, administration of substances like magnesium sulfate, statins, fasudil hydrochloride, erythropoietin, endothelin-1 antagonists, nitric oxide progenitors, and sildenafil (Siasios et al. 2013). Despite recent advances in treatment strategies, vascular microsurgery and endovascular techniques, it has been estimated that up to 15% of patients surviving the ictus of subarachnoid haemorrhage experienced stroke or death from vasospasm (Komotar et al. 2008). Therefore, it is mandatory to search for a more effective treatment to prevent such outcome.

Danshen and Gegen are commonly used in combination for treatment of coronary heart diseases (Zhang et al. 1991; Chen 2002; Ren and Luo 2006). The use of this combined drug therapy was supported by studies that showed many of the ingredients of these two herbs have cardioprotective and anti-atherosclerotic effects (Adams et al. 2006; Lam et al. 2006b, 2007, 2008a,b; Deng et al. 2012; Cheung et al. 2012). Information of Danshen and Gegen in the cerebral vascular is scant. Therefore, we have investigated the individual and combined effects of Danshen and Gegen on rat cerebral basilar arteries. A 3D-HPLC chromatogram of the DG decoction (ratio 7:3) used in the present studies is shown in Fig. 1.

Individual and combined effects of Danshen and Gegen on U46619-precontracted tone

Danshen and Gegen administered individually or as a combined DG decoction, produced relaxation of the 1146619-precontracted tone with similar [IC.sub.50] values: 0.895 [+ or -] 0.121 mg/ml for Danshen, 0.952 [+ or -] 0.139mg/ml for Gegen, and 1.001 [+ or -] 0.001 mg/ml for DG (Fig. 2A-C). The Cl values calculated at their [ED.sub.25], [ED.sub.50], and [ED.sub.75] were 1.222,1.041, and 0.902, respectively (Fig. 2D). These Cl values indicate the combined effects of Danshen and Gegen were antagonistic at low doses (e.g. [ED.sub.25]) but were additive at higher doses (e.g. [ED.sub.50] and [ED.sub.75]). By contrast, previous reports suggest their combined use can evoke synergistic effect in suppressing contraction of rat detrusor smooth muscles and in reducing LPS-induce nitric oxide (NO) production in RAW264.7 cells (Liang et al. 2012; Cheung et al. 2012). On the other hand, their combined use produced additive inhibition on foam cell formation in RAW264.7 cells, but antagonistic efficacy in suppressing proliferation of A7r5 vascular smooth muscle cells (VSMC) (Cheung et al. 2012). Overall, the accumulated evidence suggests the combined use of Danshen and Gegen can elicit vasodilator effects, and inhibits inflammation, foam cell formation, and VSMC proliferation. These data support the use of DG in atherosclerotic diseases, even though the latter effect was less than the additive effects of the two parent herbs.

Endothelium-dependent and independent mechanisms

Vasodilatation can be produced by several mechanisms that may or may not involve endothelium derived relaxant factors (EDRF). In the present study, DG, Danshen and Gegen produced identical vasodilator effects in endothelium-intact and endothelium-denuded arteries (Fig. 2A-C). This indicates an absence of a role for EDRF in their effects. Other major mechanisms of vasodilatation include activation of the guanylyl cyclase (GC)/cGMP system, and activation of the adenylyl cyclase (AC)/cAMP system (Rembold 1992). These mechanisms were examined by testing the effects of the GC inhibitor ODQ and the AC inhibitor SQ22536 on the vasodilator responses to DG, Danshen and Gegen. Neither of these inhibitors produced a change on their vasodilator effects (Tables 1-3). This ruled out the participation of GC/cAMP- and AC/cGMP-dependent pathways.

Cerebral blood vessels are known to possess multiple types of [K.sup.+] channels (Faraci and Fleistad 1998). When [K.sup.+] channels are opened, efflux of [K.sup.+] occurs and this causes hyperpolarization of cell membranes, which in turn closes voltage-dependent [Ca.sup.2+] channels and results in VSMC relaxation (Kitazono et al. 1995). It is suspected that the cerebral vasodilator effects of DG, Danshen and Gegen could be mediated by this mechanism. Therefore, several [K.sup.+] channel inhibitors including a BKCa inhibitor iberiotoxin, a [K.sub.V] channel inhibitor 4-aminopyridine, and a [K.sub.IR] channel inhibitor barium chloride were tested on the vasodilator responses to DG, Danshen and Gegen. However, none of these inhibitors produced significant change on their vasodilator effects (Tables 1-3). This discarded a role for the [BK.sub.Ca], [K.sub.V], and [K.sub.IR] channels. Previously, we have performed similar studies on a few isoflavonoids contained in Gegen in the same rat basilar artery preparations. The vasodilator effects of daidzein and daidzin were also shown to be independent of EDRF, GC/cGMP, AC/cAMP, [BK.sub.Ca], [K.sub.V], and [K.sub.IR] channels (Deng et al. 2012). However, another isoflavonoid puerarin was found to relax the rat basilar artery partly dependent on the production of endotheliumderived nitric oxide (Deng et al. 2012). Hitherto, there is no report on the cerebral vascular effects of Danshen constituents. Nonetheless, studies on rat coronary arteries showed that several Danshen constituents including danshensu, salvianolic acid B, dihydrotanshinone and cryptotanshinone, produced vasodilator effects that did not involve EDRF, or the AC/cAMP and GC/cGMP pathways (Lam et al. 2006b, 2007, 2008a,b). In addition, salvianolic acid B was shown to activate [BK.sub.Ca] channels in porcine coronary smooth muscle cells (Lam et al. 2006a).

To further elucidate a role for [K.sup.+] channels in mediating the vasodilator effects of DG, Danshen and Gegen, we have examined their effects in the presence of a [K.sub.ATP] channel inhibitor glibenclamide, a non-selective [K.sup.+] channel inhibitor TEA, and a combination of ail the [K.sup.+] channel inhibitors (i.e., iberiotoxin + 4-aminopyridine +barium chloride + glibenclamide + TEA). These treatments suppressed the maximum vasodilator effect (Imax) of DG by 21, 46 and 39%, the Imax of Danshen by 22, 35 and 34%, and the Imax of Gegen by 35, 42 and 49%, respectively (Tables 1-3). Thus, [K.sub.ATP] channels seem to play a part in mediating their vasodilator effects.

Electrophysiological studies on [K.sub.ATP] channels

To reinforce the above pharmacological studies that demonstrated involvement of [K.sub.ATP] channels in the vasodilator effects of DG, Danshen, and Gegen, electrophysiological studies were performed to examine their effects on [K.sub.ATP] channels of VSMC isolated from rat basilar arteries. Application of cromakalim caused an enhancement of the VSMC current which was sensitive to inhibition by an ATP-dependent [K.sup.+] channel blocker glibenclamide (Fig. 3A). This confirmed that the current being recorded was the genuine [K.sub.ATP] channel current. Application of DG caused a concentration-dependent enhancement of this [K.sub.ATP] current; at [less than or equal to] 0.7 mg/ml, it had no effect on the [K.sub.ATP] current amplitude, but at 1 mg/ml and 3 mg/ml, it increased the [K.sub.ATP] current amplitude (Fig. 3B). Similarly, applications of 1 mg/ml and 3 mg/ml of Danshen or Gegen produced enhancement of the [K.sub.ATP] current amplitude (Fig. 4). Therefore, DG, Danshen and Gegen were shown to be capable of producing concentration-dependent enhancement of [K.sub.ATP] current amplitudes, which support a role of the [K.sub.ATP] channels in mediating their vasodilator effects.

Pharmacological and confocal studies on [Ca.sup.2+] channels

Many blockers of voltage-operated [Ca.sup.2+] channels are effective vasodilators because they inhibit the influx of extracellular [Ca.sup.2+] into VSMC, which is important for muscle contraction (Nelson 2010). Such mechanism of action may also apply to the vasodilator effects of DG, Danshen and Gegen. To test this idea, pharmacological studies were performed on basilar artery rings bathed in [Ca.sup.2+]-free buffer primed with 100nM of U46619. Re-introducing Ca[Cl.sub.2] into the preparation elicited vasoconstriction, which was abrogated by nifedipine, a typical L-type voltage-operated [Ca.sup.2+] channel blocker (Fig. 5A). This confirmed the importance of influx of [Ca.sup.2+] via L-type [Ca.sup.2+] channels in the contractile response to U46619. Moreover, treatment with DG, Danshen and Gegen inhibited the Ca[Cl.sub.2]-induced contraction in a concentration-dependent manner, indicating that these agents can prevent [Ca.sup.2+] influx in the preparation (Fig. 5A-C).

To corroborate these observations, confocal microscopic studies were performed on [Ca.sup.2+] influx in VSMC isolated from rat basilar arteries. As shown in Fig. 6, restoration of extracellular [Ca.sup.2+] (2.5 mM) in VSMC after [Ca.sup.2+] store depletion led to a rapid increase in [[Ca.sup.2+].sub.i] that peaked at 4s. The magnitude of the fluorescence increase was about 600% relative to the resting state and the area under the curve ([AUC.sub.0-100s]) value was 520.8 [+ or -] 76.0. In the presence of DG (3 mg/ml), Danshen (3 mg/ml) or Gegen (3 mg/ml), [Ca.sup.2+] application produced [AUC.sub.0-100s] values of 136.5 [+ or -] 23.4,83.8 [+ or -] 24.8, and 126.1 [+ or -] 39.2, representing 74%, 84% and 76% inhibition on the increase in [[Ca.sup.2+].sub.i] respectively. Nifedipine (100nM) also attenuated the increase in [[Ca.sup.2+].sub.i]. with an [AUC.sub.0-100s] value of 249.9 [+ or -] 57.5, which represents 52% inhibition. These findings confirmed that inhibition of [Ca.sup.2+] influx is an important mechanism for the vasodilator effects of DG, Danshen and Gegen.

Conclusions

In the present study, Danshen and Gegen were shown to be potential alternative natural products for prevention or treatment of cerebral vasospasm, in addition to the use of conventional calcium channel blockers. Used either alone or together, these two herbs produced prominent vasodilator effects on rat basilar arteries. Moreover, their vasodilator effects can be additive when used as a combined decoction. Pharmacological, electrophysiological and confocal microscopic studies confirmed their mechanisms of vasodilatation involved opening of [K.sub.ATP] channels and inhibition of [Ca.sup.2+] influx in VSMC. Further studies are currently underway to elucidate the cerebral protective efficacies of their two herbs in animal models of stroke.

Conflict of interest

The authors have no conflict to disclose.

ARTICLE INFO

Article history:

Received 20 June 2013

Received in revised form 7 August 2013

Accepted 29 September 2013

Acknowledgements

The work described in this paper was supported by a grant from University Grant Committee Area of Excellence project "Chinese Medicine Research and Further Development" of the Hong Kong Special Administrative Region, China (Project No. AoE/B-10/01). Provision of a post-graduate studentship to Miss Y. Deng by the Chinese University of Hong Kong is acknowledged.

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Y. Deng (3), E.S.K. Ng (a), Y.W. Kwan (a), C.B.S. Lau (b,c), D.W.S. Cheung (b,c), J.C.M. Koon (b,c), Z. Zhang (d), Z. Zuo (d), P.C. Leung (b,c), K.P. Fung (a,b,c), F.F.Y. Lam (a,*)

(a) School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region

(b) Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region

(c) State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region

(d) School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region

* Corresponding author at: Room 226, Lo Kwee Seong Integrated Biomedical Sciences Building, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region. Tel.: +852 3943 6790; fax: +852 2603 5123.

E-mail address: francisLam@cuhk.edu.hk (F.F.Y. Lam).

http://dx.doi.org/10.1016/j.phymed.2013.09.016

Table 1 Effects of various treatments on the relaxant action of water
extract of a combined Danshen and Gegen (DG) formulation on U46619-
precontracted tone in rat isolated basilar arteries.

Treatments                    n     [IC.sub.50] [+ or -]
                                    SEM (mg/ml)

Control                       7     0.875 [+ or -] 0.049
+100 [micro]M SQ22536         7     0.847 [+ or -] 0.089
Control                       6     0.835 [+ or -] 0.158
+100 [micro]M ODQ             6     0.916 [+ or -] 0.101
Control                       6     0.902 [+ or -] 0.011
+100 [micro]M iberiotoxin     6     0.863 [+ or -] 0.062
Control                       6     0.995 [+ or -] 0.010
+1 [micro]M                   6     0.902 [+ or -] 0.206
4-aminopyridine
Control                       6     0.914 [+ or -] 0.227
+100 [micro]M barium          6     1.174 [+ or -] 0.211
chloride
Control                       6     1.062 [+ or -] 0.063
+1 [micro]M glibenclamide     6     0.956 [+ or -] 0.074
Control                       6     1.212 [+ or -] 0.089
100 [micro]M TEA              6     2.604 [+ or -] 0.574 **
Control                       6     1.260 [+ or -] 0.212
+A combination of [K.sup.*]   6     1.223 [+ or -] 0.052
channel inhibitors

Treatments                    Imax [+ or -] SEM (%)

Control                       -101.100 [+ or -] 5.213
+100 [micro]M SQ22536         -97.750  [+ or -] 3.739
Control                       -115.300 [+ or -] 4.949
+100 [micro]M ODQ             -107.000 [+ or -] 3.835
Control                       -97.670  [+ or -] 15.440
+100 [micro]M iberiotoxin     -86.420  [+ or -] 12.720
Control                       -97.470  [+ or -] 9.837
+1 [micro]M                   -98.130  [+ or -] 9.489
4-aminopyridine
Control                       -111.200 [+ or -] 3.852
+100 [micro]M barium          -97.660  [+ or -] 2.706
chloride
Control                       -114.900 [+ or -] 5.370
+1 [micro]M glibenclamide     -90.530  [+ or -] 2.616 **
Control                       -112.100 [+ or -] 2.943
100 [micro]M TEA              -60.020  [+ or -] 10.350 **
Control                       -108.200 [+ or -] 11.420
+A combination of [K.sup.*]   -66.320  [+ or -] 12.000 *
channel inhibitors

Glibenclamide, TEA, or a combination of [K.sup.*] channel inhibitors
(iberiotoxin + 4-aminopyridine +barium chloride + glibenclamide+TEA)
produced significant inhibition on the DG-induced vasodilator effect.
[IC.sub.50] values indicate the drug concentrations that produced 50%
inhibition on the U44619-precontracted tone, and negative Imax values
indicate the maximum percentages of inhibition. * p<0.05, ** p<0.01.

Table 2 Effects of various treatments on the relaxant action of water
extract of Danshen on U46619-precontracted tone in rat isolated
basilar arteries.

Treatments                    n    [IC.sub.50] [+ or -]
                                   SEM (mg/ml)

Control                       6    1.039 [+ or -] 0.208
+100 [micro]M SQ22536         6    0.786 [+ or -] 0.059
Control                       6    0.842 [+ or -] 0.095
+100 [micro]M ODQ             6    0.975 [+ or -] 0.245
Control                       6    1.031 [+ or -] 0.114
+100 [micro]M iberiotoxin     6    0.903 [+ or -] 0.038
Control                       7    1.370 [+ or -] 0.335
+1 mM                         7    0.814 [+ or -] 0.041
4-aminopyridine
Control                       5    1.242 [+ or -] 0.135
+100 [micro]M barium          5    1.290 [+ or -] 0.109
chloride
Control                       7    1.118 [+ or -] 0.729
+1 [micro]M glibenclamide     7    1.057 [+ or -] 0.144
Control                       6    0.991 [+ or -] 0.006
100 mM TEA                    6    3.982 [+ or -] 1.022 **
Control                       6    0.973 [+ or -] 0.064
+A combination of [K.sup.*]   6    3.891 [+ or -] 1.640 **
channel inhibitors

Treatments                    Imax [+ or -] SEM (%)

Control                       -100.550 [+ or -] 5.842
+100 [micro]M SQ22536         -110.729 [+ or -] 5.052
Control                       -111.462 [+ or -] 4.600
+100 [micro]M ODQ             -102.184 [+ or -] 3.204
Control                       -114.015 [+ or -] 3.208
+100 [micro]M iberiotoxin     -108.887 [+ or -] 6.140
Control                       -103.708 [+ or -] 3.927
+1 mM                         -98.987  [+ or -] 6.712
4-aminopyridine
Control                       -108.961 [+ or -] 6.712
+100 [micro]M barium          -93.288  [+ or -] 6.961
chloride
Control                       -115.128 [+ or -] 2.543
+1 [micro]M glibenclamide     -90.140  [+ or -] 2.889 **
Control                       -108.472 [+ or -] 5.058
100 mM TEA                    -70.715  [+ or -] 6.128 **
Control                       -115.858 [+ or -] 7.048
+A combination of [K.sup.*]   -76.929  [+ or -] 4.860 **
channel inhibitors

Glibenclamide, TEA, or a combination of [K.sup.*] channel inhibitors
(iberiotoxin+ 4-aminopyridine + barium chloride + glibenclamide +
TEA) produced significant inhibition on the Danshen-induced
vasodilator effect. [IC.sub.50] values indicate the drug
concentrations that produced 50% inhibition on the U44619-
precontracted tone, and negative Imax values indicate the maximum
percentages of inhibition, ** p < 0.01.

Table 3 Effects of various treatments on the relaxant action of water
extract of Gegen on U46619-precontracted tone in rat isolated basilar
arteries.

Treatments                    n     [IC.sub.50] SEM (mg/ml)

Control                       6     0.803 [+ or -] 0.033
+100 [micro]M SQ22536         6     0.829 [+ or -] 0.092
Control                       6     0.981 [+ or -] 0.160
+100 [micro]M ODQ             6     0.731 [+ or -] 0.057
Control                       6     0.990 [+ or -] 0.124
+100 [micro]M iberiotoxin     6     1.104 [+ or -] 0.076
Control                       6     0.746 [+ or -] 0.066
+1 [micro]M                   6     0.670 [+ or -] 0.055
4-aminopyridine
Control                       5     0.906 [+ or -] 0.143
+100 [micro]M barium          5     1.025 [+ or -] 0.038
chloride
Control                       7     0.838 [+ or -] 0.196
+1 [micro]M glibenclamide     7     0.945 [+ or -] 0.110
Control                       6     0.890 [+ or -] 0.006
100 mM TEA                    6     1.373 [+ or -] 0.017 *
Control                       6     0.856 [+ or -] 0.118
+A combination of [K.sup.*]   6     0.992 [+ or -] 0.178
channel inhibitors

Treatments                    /mox[+ or -]SEM (%)

Control                       -106.467 [+ or -] 5.019
+100 [micro]M SQ22536         -107.322 [+ or -] 8.497
Control                       -114.843 [+ or -] 5.957
+100 [micro]M ODQ             -110.960 [+ or -] 6.384
Control                       -110.054 [+ or -] 3.313
+100 [micro]M iberiotoxin     -95.202  [+ or -] 2.905
Control                       -106.126 [+ or -] 3.954
+1 [micro]M                   -100.117 [+ or -] 6.442
4-aminopyridine
Control                       -107.381 [+ or -] 3.084
+100 [micro]M barium          -94.917  [+ or -] 3.173
chloride
Control                       -116.41  [+ or -] 3.887
+1 [micro]M glibenclamide     -75.330  [+ or -] 6.257 ***
Control                       -112.584 [+ or -] 4.174
100 mM TEA                    -64.769  [+ or -] 4.860 **
Control                       -116.043 [+ or -] 3.483
+A combination of [K.sup.*]   -59.344  [+ or -] 6.159 **
channel inhibitors

Glibenclamide, TEA, or a combination of [K.sup.*] channel inhibitors
(iberiotoxin+4-aminopyridine +barium chloride + glibenclamide + TEA)
produced significant inhibition on the Gegen-induced vasodilator
effect. [IC.sub.50] values indicate the drug concentrations that
produced 50% inhibition on the U44619-precontracted tone, and
negative Imax values indicate the maximum percentages of inhibition.
* p<0.05, ** p<0.01, *** p<0.001.
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Author:Deng, Y.; Ng, E.S.K.; Kwan, Y.W.; Lau, C.B.S.; Cheung, D.W.S.; Koon, J.C.M. J.C.M.; Zhang, Z.; Zuo,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Mar 15, 2014
Words:5552
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