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Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum.

ARTICLE INFO

Keywords: Schisandra chinensis Lignans Smooth muscle relaxation Calcium channels

ABSTRACT

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC20 values ranging from 2.2[+ or -]0.4 [micro] M (schisandrin A) to 13.2[+ or -]4.7 [micro] M (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In [Ca.sup.2+] -free preparations primed with ACh or KCl, schisandrin A (50 [micro] M) attenuated the contractile responses to cumulative addition of Ca [Cl.sub.2] by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in [Ca.sup.2+] free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both [Ca.sup.2+] influx through L-type [Ca.sup.2+] channels and intracellular [Ca.sup.2+] mobilization, rather than specific antagonism of cholinergic muscarinic receptors.

[c] 2011 Elsevier GmbH. All rights reserved.

Introduction

Coordination of smooth muscle contraction and relaxation is the basis of gut motility, which is regulated by enteric nervous system via a number of excitatory and inhibitory mediators (Schemann 2005). In most parts of the gastrointestinal (GI) tract, acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5-HT) are the important neurotransmitters which initiate excitatory events (e.g. spasms and visceral pain) in response to mechanical or chemical stimuli (Gershon and Tack 2007; Tobin et al. 2009). Cholinergic antagonism is a conventional approach to produce antispasmodic effect for treating diarrhoea. Substances with abilities to antagonize 5-HT-mediated events may slow small bowel and colonic transit, as well as reduce colonic tonic response to feeding and sensation of volume of distensions (De Ponti and Malagelada 1998; Bueno 2005).

Schisandra Chinensis (Turcz.) Baill (Schisandraceae) is widely found in the northwest part of China, Korea, and the far east of Russia (Panossian and Wikman 2008). In traditional Chinese medicine (TCM), the dried ripe fruits, named "wu-wei-zi" (five taste fruit), are indexed as a tonic, astringent and sedative agent in Chinese Pharmacopoeia (National Committee of Chinese Pharmacopoeia 2010). As demonstrated by a number of investigations, dibenzo[a, c]cyclooctadiene lignans, the main chemical components in S. chinensis, are generally considered as the major active ingredients which contribute to the anti-oxidant, hepatic detoxifying, neuroprotective, and anti-cancer activities of this crude drug (Lu and Liu 1992; Zhu et a1.1999; Kim etal. 2004; Huang et al. 2008; Panossian and Wikman 2008). S. chinensis extracts and lignans have also shown relaxant effects on isolated smooth muscles, including trachea, mesenteric arteries and thoracic aorta, and some of these actions were claimed to involve direct effects on smooth muscles, such as blockade of [Ca.sup.2+] influx (Suekawa et al. 1987; Rhyu etal. 2006; Park etal. 2009). However, their effects on GI motility are far from understood, in spite of the use of this herb for treating protracted diarrhoea in clinical trials in children suffering from dysentery (Zuzanova and Bakhtina 1954) and in the treatment of acute enterocolitis with clinical symptoms typical of light and mild forms of acute dysentery (n = 400) (Pelishenko 1972). The considerable diversity of the pharmacological effects of Schisandra chinensis in numerous studies over 40 years have recently been reviewed and concluded that the lignans may be useful as a source from which new drugs may be discovered (Panossian and Wikman 2008).

In our previous study, S. chinensis extracts and the major lignans have been demonstrated to cause inhibitions on the spontaneous contractions of rat colon (unpublished data). The action of schisandrin, one of the major lignans, was likely mediated by non-cholinergic non-adrenergic transmitter(s) released from enteric nerve (Yang et al. 2011). This study aims to evaluate the modulatory effects of S. chinensis on smooth muscles of isolated guinea pig ileum, determine the corresponding active ingredients and investigate the relevant mechanism(s) of the actions. The antispasmodic effects were examined by the actions against the contractile responses to ACh and 5-HT. The contents of four major lignans in Schisandra extracts, schisandrin, schisandrol B, schisandrin A and schisandrin B, were quantified by HPLC, and their effects on intestinal contractions were further determined using the same bioassays. Furthermore, some of the relevant mediators involved in the action of schisandrin A were identified using receptor binding assays and ion channel blockers.

Materials and methods

Chemicals and reagents

Dried berries of Schisandra chinesis were provided by Zhixin Pharmaceutical Company (Guangdong, China), identified and authenticated by Ms. Yuying Zong, School of Chinese Medicine, The Chinese University of Hong Kong. Schisandrin, schisandrin A and schisandrin B were obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and schisandrol B was from the Hong Kong Jockey Club Institute of Chinese Medicine. HPLC grade acetonitrile (Fisher, USA) and trifluoroacetic acid were used for chromatography. Water was purified by Milli-Q. academic purification system (Millipore, USA). Acetylcholine (ACh), 5-hydroxytryptamine (5-HT), atropine, tropisetron and nifedipine were purchased from Sigma Co. (USA). The chemicals for buffer solution were analytical grade for common laboratory use. Stock solution of the aqueous extract of S. chinesis was prepared with distilled water and diluted with saline before use, whereas the ethanol extract and Schisandra lignans were dissolved in 100% dimethyl sulfoxide (DMSO) as stock and diluted with DMSO. All stock solutions were stored at -20[degrees]C Each drug administration was between 0.5 and 30 [micro] l, and thus in each experiment, the final DMSO concentration did not exceed 0.3%.

Preparation of Schisandra extracts

Dried S. chinensis fruits (500g) were soaked in 41 of distilled water for 30 min at room temperature. Then, the fruits were boiled three times for 1 h with 41, 31 and 31 of distilled water, successively. The combined decoction was filtered and condensed by rotor evaporation under reduced pressure. Finally, the concentrate was freeze-dried to obtain the aqueous extract powder with the yield of 35.2%. Ethanol extract was obtained by 95% ethanol extraction following the same procedures described above and the extract yield was 27.6%. The dry powder was placed in a desiccator at room temperature until use. For quantitative determination, plant extracts were sonicated in 50-fold methanol for 30 min, followed by centrifugation and filtration, prior to HPLC analysis.

HPLC conditions and quantification of Schisandra lignans

The samples were separated by HPLC at a flow rate of 0.8 ml/min. The mobile phase consisted of 0.1% trifluoroacetic acid water (A) and acetonitrile (B) with a gradient elution of 20% B at 0-5 min; 45% B at 6-35 min; 45-60% at 35-50 min; 60-100% B at 50-60 min. The column temperature was 25[degrees]C The injection volume was 20 [micro] l and the detection wavelength was set at 230 nm. Simultaneous quantification of schisandrin, schisandrol B, schisandrin A and schisandrin B was performed on six levels of external standards.

The calibration curves showed good linear correlation ([r.sup.2] [greater than or equal to] 0.9995) between peak area and concentration. Intra- and inter-day precision of the method was within the acceptable limits with R.S.D. < 5%. Samples were stable for 72 h at room temperature (R.S.D. < 3%). The recovery of each analyte ranged from 96.5% to 108.9%.

Animals

Male guinea pigs (350-500 g) were bred and housed by the Laboratory Animal Services Centre of the Chinese University of Hong Kong. All experiments were performed with approval from the Animal Research Ethics Committee, The Chinese University of Hong Kong. The animals were kept in a temperature controlled room (23 + 2[degrees]C ) with a 12-h light-dark cycle, with free access to food and water.

Effects on acetylcholine (ACh) - or 5-hydroxytryptamine (S-HT)-induced contraction in guinea pig ileum

Male guinea pigs were fasted overnight and sacrificed by cervical dislocation and exsanguination. A 20 cm length of ileum was removed and placed in Krebs-Henseleit solution: NaCI 118, KC1 4.7, Ca [Cl.sub.2] 2.5, Mg [SO.sub.4] 1.2, K [H.sub.2] [P0.sub.4] 1.18, NaH [C0.sub.3] 25, and glucose 10 mM. After cleaning of mesenteric tissues, the ileum was cut into segments of 2 cm length and suspended in 10 ml organ bath filled with Krebs solution. The bathing solution was maintained at constant temperature 37[degrees] C and aerated with 95%[0.sub.2]/5%[C0.sub.2]. Washout of the organ bath was performed by solution upward displacement and overflow. Contractions of the longitudinal muscle of guinea-pig ileum were measured with Grass FT03 isometric transducers connected to Powerlab data acquisition system (ADInstruments Pty Ltd., Australia). An initial tension of 0.8 g was applied. After equilibration for about 1 h, with frequent washes, the resting tension remained steady at approximately 0.4g. An appropriate agonist (ACh or 5-HT) at the final concentration of 10 [micro] M was tested on each preparation to ensure a steady and acceptable level of tension (about 3.0 g) had been reached before the experimental procedures began. After washout with Krebs solution, the isolated ileum was allowed to rest for 7 min and 10 [1.M ACh or 5-HT was added again to produce reference tone. Concentration-response curves of Schisandra extracts and Schisandra lignans were obtained by non-cumulative pre-incubation of these inhibitory agonists for 15 min before addition of ACh or 5-HT. After 3 min treatment, a standard procedure with washing for 3 min and resting for 7 min was allowed between each drug administration.

Studies on the involvement of [Ca.sup.2+] channels

The isolated ileums were equilibrated in [Ca.sup.2+] -free Krebs solution containing 0.2 mM [Na.sub.2] -EGTA for 60 min and washed three times at 20 min intervals. ACh (10 [micro] M) or KCI (60 mM) was added as a primer to depolarize the plasma membrane. Afterwards, cumulative dose of Ca [Cl.sub.2] (0.01-4.44mM) was added at 6min intervals to produce concentration-response contraction. When the maximum contractile tension was achieved, the tissues were washed out and equilibrated for 30min with three-time washing at 10 min intervals. Then the ileum strips were pre-incubated with 0.1% DMSO (vehicle control), schisandrin A (50 [micro] M) or nifedipine (300 nM) for 15 min. Subsequently priming with ACh or KC1, the concentration-dependent contractile responses to cumulative addition of Ca [Cl.sub.2] were repeated to determine the inhibitory effects of test drugs on the contractions elicited by [Ca.sup.2+] influx.

Studies on the involvement of intracellular [Ca.sup.2+]

In another set of experiments to investigate the involvement of intracellular [Ca.sup.2+], the ileum strips were equilibrated in [Ca.sup.2+] -free solution without addition of [Na.sub.2] -EGTA. ACh (10 [micro] M) was first added to produce a reference tension. The tissues were then washed twice with normal Krebs solution at 15 intervals for refilling the intracellular [Ca.sup.2+] store, followed by twice washing with [Ca.sup.2+] -free buffer and equilibration for 30min. Then the second contractile responses to ACh were tested after pre-incubation with DMSO (vehicle control) or schisandrin A (20-100 [micro] M) for 15min.

Measurement of receptor binding ofSchisandra lignans

Binding of Schisandra lignans to cholinergic muscarinic (M3) receptor was analyzed by a commercially available cell-based assay in GeneBLAzer M3 CHO-K1 DA cells according to the manufacturer's notes (Invitrogen, Carlsbad, CA). The receptor binding was evaluated by the dual-color (blue/green) fluorescence ratio with carbachol as an agonist. Effects of schisandrin, schisandrin A and schisandrin B were compared to DMSO (vehicle blank), and atropine was used as a positive control. Schisandrol B was not included due to the unavailability of the reference compound during the course of this assay.

Data analysis

Inhibitory responses of Schisandra extracts and lignans on the contractility of isolated guinea pig ileum were measured as the reduction of maximum tension (in grams over baseline) induced by the corresponding agonists (ACh or 5-HT). Values were taken as the percentage change from reference tension (e.g.--100% corresponds to the abolition of contraction). All values were expressed as mean [+ or -] S.E.M., where n was the number of preparations from different animals used. GraphPad Prism software was used to fit sig-moidal curves (variable slope) and to determine the [pounds sterling] max and [EC.sub.50] or ([EC.sub.20]) values from the concentration-response curves. Results from the controls and treatments were compared by unpaired oneway or two-way ANOVA followed by Dunnett post test. All tests were two-tailed and the significance was set at P < 0.05.

Results

HPLC characterization of Schisandra extracts

Fig. 1 shows that all four lignans were well separated in a mixture of standards as well as in Schisandra extracts. Determined by the validated method, the quantities of four Schisandra lignans in aqueous extract and 95% ethanol extracts were shown in Table 1. Schisandrin was found to be the most abundant lignans, covering 75.4% and 46.6% of total amount of the analyzed lignans in aqueous extract and 95% ethanol extracts, respectively. In aqueous extract, the amounts of schisandrin (2960 [+ or -] 28 [micro] g/g) and schisandrol B (852 [+ or -] 16 [micro] g/g) were considerable while only trace amounts of schisandrin A (49[+ or -]2 [micro] g/g) and schisandrin B (60[+ or -]2 [micro] g/g). In the ethanol extract, higher contents of all lignan compounds were Found. Schisandrin and schisandrol B were increased by 4-8 folds, whereas the increases were 50-fold and 190-fold for schisandrin A and schisandrin B, respectively. This observation agreed with the lipophilicity of schisandrin, schisandrol B, schisandrin A and schisandrin B. The total contents of four analyzed lignans found in the aqueous and ethanol extracts were 3.9 and 40mg/g, respectively, approximately in the proportion of 1:10.4.
Table 1
Amounts of four lignans present in the aqueous extract and 95%ethanol
extract of Schisandra chinensis fruits (n = 5).

Analyte      Aqueous extract            95% Ethanol
                                            extract

              Mean [+ or -]      R.S.D.    Mean[+ or -]     R.S.D.
              S.D.([mu]g/g)      (%)S.D.([mu]g/g)            (%)

Schisandrin   2.960 [+ or -]    0.96  19.024 [+ or -]    1.45
                          28                      275

Schisandrol  852 [+ or -] 16    1.86    7.501[+ or -]    2.01
B                                                 151

Schisandrin    49 [+ or -] 2    3.77   2.684 [+ or -]    1.30
A                                                  35

Schisandrin    60 [+ or -] 2    2.83  11.588 [+ or -]    1.52
B                                                 177


Effects on ACh- and 5-HT-induced cont ractions inguinea pig ileum

As shown in Fig. 2(a ), both aqueous and ethanol extracts of S. chinensis fruits produced concentration-de pendent inhibitions on ACh-induced contraction of isolated guinea pig ileum. but the ethanol extract was nearl y 20 times more potent than aqueous extract [EC.sub.20]: 60 [+ or -] 11 vs. 1182 [+ or -] 355, P < 0.00l, Table 2). The Jignans, schisa ndrin, schisandrol B, schisandrin Aand schisandrin B, also caused concentration-dependent inhibitions in ACh-induced contraction. whi le DMSO (vehicle blank ) had no significant effect (Fig. 2(b)). Schisandrin Awas the most potent. followe d by schisandrin B. schisandrol B and schisa ndri n. with their E[C.sub.20] values of 2.2 [+ or -] 0.4. 3.1 [+ or -] 0.6. 3.5 [+ or -]0.7, 13.2 [+ or -] 4.7 [micro]M, and the [E.sub.max] values of - 94.5 [+ or -] 6.5%. - 98.3 [+ or -] 13.6%. - 47.0 [+ or -] 3,9%, - 34,6 [+ or -] 5.1%, respectively (Table 2).At ropine concen t ration-dependently inhibited the cont ractile responses. resulting in an E[C.sub.50] value of 25.0 [+ or -] 0.3 [micro]M and an abolishment (> 90%) at the concent rat ion of 1 [micro]M. Additionally the Emax value of at ropine (-94.6 [+ or -] 2.0%) was comparable to schisandrin Aand schisa nd rin Bbut markedly higher than schisandrin and schisandrol B.
Table 2 E [C.sub.20] or E [C.sub.50] of Schisandrct extracts and the
major lignans in relaxing Ach- or 5-HT-inducecl contraction isolated
guinea pig ileum. Values are expressed as mean [+ or -] S.E.M (n=5-6).

Substances       ACh induced         5-HT induced
                contraction         contraction
                E [C.sub.20]  [E.sub.max]  E [C.sub.50]  [E.sub.max]
               ([mu]g/g)          (%)      ([mu]g/g)        (%)

Aqueous          1.182 [+ or  -43.0 [+ or       > 5000    -44.9 [+ or
extract               -] 355       -] 6.4                     -] 8.1%

Ethanol          60 [+ or -]  -91.3 [+ or  148 [+ or -]   -95.3 [+ or
extract                   11      -] 16.6            28       -] 6.6%

Schisandrin       13.2 [+ or  -34.6 [+ or    40.7 [+ or   -64.1 [+ or
                      -] 4.7       -] 5.1        -] 5.4        -] 6.5

Schisandrol     3.5 [+ or -]  -47.0 [+ or    24.5 [+ or   -61.0 [+ or
B                        0.7       -] 3.9        -] 2.2        -] 7.7

Schisandrin     2.2 [+ or -]  -94.5 [+ or  6.6 [+ or -]   -84.1 [+ or
A                        0.4       -] 6.5           0.6        -] 6.3

Schisandrin     3.1 [+ or -]  -98.3 [+ or    10.0 [+ or   -83.6 [+ or
B                        0.6      -] 13.6        -] 0.8        -] 7.2


The relaxant effects of Schisandra extracts and lignans were also determined using another contractile agon ist, 5-HT. As shown in Fig. 3(a), the ethanol extract caused a concen trat ion-de pendent inhibition on 5-HT-induced contraction, whereas the aqueous extrac t only produced significant responses (-44.9 [+ or -] 8. 1%) at the conce ntra tion of 5 mg/ml. As indica ted by the ECsovalues in Table 2, the effectiveness of the ethanol extract was over 30 times greater than the aqueous extract. Fig. 3(b) shows that all four Schisandra Iignans relaxed 5-HT-induced cont ract ions in a conce ntrationdependent manner. Schisandrin A was the most potent followed by schisandrin B. schisandrol B and schisandrin with th eir E[C.sub.50] value s and Emax values summarized in Table 2. Tropisetron, a selective 5-H[T.sub.3] receptor an tagonist inhibited 5-HT-induced contraction. producing maximum inhibi tion (- 54.7 [+ or -] 6.1%) at 1 tLM and E[C.sub.50] at 0.18 [+ or -]0.01 [micro]M.

Binding ofSchisandra lignam to cholinergic muscarinic (M3) receptor

To determine the mechani sm(s) of antispasmodic effects of Schisandra lignans, receptor binding assays for cholinergic muscarinic (M3) receptor were performed in M3 CHO-Kl DA cells. Carbachol, an agonist of muscarinic receptor caused concentration-dependent activation on the fluorescence responses (blue/green). The possible antagonistic effects of te st drugs were examined by the inhibition on carbachol-induced fluorescence activation. The more inhibi tion the drug exhibits. the higher affinity and potency of receptor binding it should process. Atropine a muscarinic receptor antago nist as a posit ive control, in hibited the fluorescence activation rat io ind uced by 2 [micro]M carbac hol in a concent ration-de pende nt manner, and abolished it at the concent rat ion of 1 [micro]M. In contrast, as shown in Fig. 4, schisandr in, schisandrin A and schisan drin B did not produce significant inhibitory effects on the fluorescence respo nse at the concentrations from 1 [micro]M to 25 [micro]M as compared to vehicle control. The inhibitory effect of 100 [micro]M schisandrin B may be related to the reduction of cell viability or interruption of fluore scence excitation. as indicated by the significant decrease s of fluorescence responses at both blue and green excitation wavelengths for about 40%.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Effect of schisandrin A on [Ca.sup.2+] -induced contraction

These experiments were performed in [Ca.sup.2+] -free buffer solution plus 0.2 mM Na2-EGTA. Priming with 10[micro]M ACh had no significant effect on the resting tone, and the tissues were subsequently treated with cumulative doses of CaCl2 (0.01-4.44 mM), which produced stepwise increase of tissue tone. As shown in Fig. 5(a), the maximum sustained tension elicited by Ca [Cl.sub.2] was 4.57[+ or -] 0.19g, and this was attenuated to 2.88[+ or -] 0.46g (37% reduction, P< 0.001) in the presence of 50 [micro]M schisandrin A, while it was essentially abolished by 300 nM nifedipine (reduction > 95%). In KCl-primed ileum strips, the maximum contraction elicited by Ca [Cl.sup.2] was 4.30 [+ or -] 0.40 g, which was attenuated to 2.71 [+ or -]0.28g (37% reduction, P < 0.001) in the presence of schisandrin A (50 [micro]M), whereas was blocked by nifedipine (300 nM) (Fig. 5(b)).

In another set of preparations in the absence of extracellular [Ca.sup.2+], ACh (10 [micro]M) was used as an agonist of intracellular [Ca.sup.2+] mobilization to produce contractile responses with the tension of 1.01 [+ or -]0.05g. As shown in Fig. 6, pre-treatment of schisandrin A (20-100 [micro] M) dose-dependently inhibited the contractile responses to ACh in [Ca.sup.2+] -free buffer, and the responses were inhibited by 78% in the presence of 100 [micro]M schisandrin A (P < 0.001).

Discussion

The present study has demonstrated that S. chinensis extracts exhibited relaxant activities on the intestinal contractility of iso-lated guinea pig ileum. The inhibitory effects of this medicinal herb on intestinal contractions appeared to be in agreement with its well-known clinical application in Chinese Medicine to control excessive loss of essential energy and body fluid, such as diarrhoea. From the corresponding effective concentrations, it would appeared that the effectiveness of S. chinensis in relaxing ACh-induced contraction approached to the documented antispasmodic activity of Magnolia officinalis, another traditional Chinese medicine commonly used for treating gastrointestinal (GI) disorders (Chan et al. 2008). S. chinensis fruits could be, therefore, considered as potential remedies for Gl diseases with diarrhoea symptoms. There remains a question about the active ingredients that are responsible for the relaxant effects of this crude drug.

Schisandra lignans are the major chemical ingredients in S. chinensis. Schisandrin, schisandrol B, schisandrin A and schisandrin B are among the most important lignans (Halstead et al. 2007). In this study, these lignans were detected in both aqueous and ethanol extract, but the contents in the ethanol extract were much higher than the aqueous extract (over 10 fold in total), especially for schisandrin A and schisandrin B (50 and 190 fold, respectively). As a result of evaluating the in vitro effects on gut motility, the four Schisandra lignans showed relaxant activities on isolated smooth muscles of guinea pig ileum with various potencies. The effectiveness of schisandrin A in relaxing the contractility induced by ACh or 5-HT was comparable, if not stronger, to schisandrin B, whereas these two compounds were markedly more effective than schisandrin and schisandrol B, based on the E [C.sub.50] (or E [C.sub.20]and [E.sub.max] values. Furthermore, the effectiveness of schisandrin A and schisandrin B was comparable to two documented antispasmodic substances magnolol and honokiol, the active ingredients of Magnolia officinalis (Chan et al. 2008). In the chemical structures, schisandrin and schisandrol B share a common feature of a 6-hydroxy substitution on the cyclooctenring, which may be related to their less potent antispasmodic activities compared to schisandrin A and schisandrin B (without 6-hydroxy substitution). Comparatively, 11, 12-methylenedioxy or methylenedioxy groups appeared to have less significant impact on the antispasmodic actions, as indicated by the comparable potencies between schisandrin and schisandrol B, or schisandrin A and schisandrin B. The four Schisandra lignans also showed similar effectiveness and structureactivity relationship in inhibiting the 5-HT-induced contractions. Moreover, the greater inhibitory potency of the ethanol extract than aqueous extract (about 20-fold) approximately matched the higher quantities of lignans contained in the ethanol extract. Therefore, in spite of the presence of other lignans in S. chinensis, the four lignans could be considered as the major active components for the antispasmodic effect of this herb.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

The present study further investigated the potential involvement of muscarinic acetylcholine receptors. In the alimentary tract, all five subtypes of the muscarinic receptors, namely M1, M2, M3, M4 and M5, can be identified (Tobin et al. 2009), but the main muscarinic receptor mediating contraction in GI smooth muscle cells is recognized to be the M3 receptor (Uchiyama and Chess-Williams 2004). Therefore a cell-based assay for measuring the binding extent to cholinergic M3 receptor was employed to determine the possible role of muscarinic receptor in the antispasmodic actions of Schisandra lignans. Atropine, a non-selective muscarinic receptor antagonist, abolished the carbachol-induced fluorescence activation at the concentration of 1 [micro]M, indicating the complete M3 receptor occupation by atropine. In contrast, schisandrin, schisan-drin A and schisandrin B had no significant effects on the fluorescent responses to carbachol except for an approximately 70% inhibition by 100|xM schisandrin B, but the effects were likely attributed to the reduction of cell viability in the presence of such a high concentration of schisandrin B.The results indicated that the antispasmodic actions of Schisandra lignans unlikely involved specific antagonism of muscarinic (M3) receptor. Nevertheless, the present results may not exclude the possible roles of other subtypes of the muscarinic receptors.

Another mechanistic study was also performed to determine the significance of [Ca.sup.2+]-dependent signalling pathway in the antispasmodic action of schisandrin A, the most potent lignan among the four compounds. Increase of intracellular [Ca.sup.2+] plays a principle role in smooth muscle contraction. In response to the external stimuli such as excitatory agonist and mechanical stretch, the intracellular concentration of [Ca.sup.2+] is elevated, and then the active [Ca.sup.2+] combines with the cytosolic protein calmodulin, which thereby activates myosin light chain (MLC) kinase to phosphorylate the light chain of myosin, resulting in smooth muscle contraction (Webb 2003). Influx of extracellular [Ca.sup.2+] through the activation of voltage-operated or receptor-operated [Ca.sup.2+] channels is an important mediator in the [Ca.sup.2+]-coupling contractile response. In our study, cumulative addition of CaCb produced concentration-dependent contractions after priming with ACh in [Ca.sup.2+] free solution containing Na2-EGTA. Nifedipine (300 nM), a selective L-type calcium channel blocker, abolished the contractions elicited by [Ca.sup.2+], indicating the involvement of L-type calcium channel in the ACh-induced ileal contraction. The maximum contractile responses to [Ca.sup.2+] were also partially inhibited by 37% in the presence of schisandrin A (50 [micro]M), which demonstrated that the antispasmodic activity of schisandrin A likely involved the blockade of receptor-operated calcium channels. In KCl-primed preparations, attenuation of [Ca.sup.2+]-induced contraction was also observed in the presence of schisandrin A, implying that this compound could block [Ca.sup.2+] influx through voltage-operated calcium channels, leading to the intestinal relaxation. Previous studies on the vasorelaxant effects of S. chinensis have shown that S. chinensis extracts, as well as an isolated lignan gomisin A (schisandrol B), caused endothelium-dependent relaxation, and also directly acted on smooth muscles, including blocking the [Ca.sup.2+] influx (Rhyu et al. 2006; Park et al. 2007, 2009).The results in this study were consistent with the previous reports showing the inhibition of Schisandra lignans on [Ca.sup.2+] influx. Therefore, the inhibitory action of schisandrin A on calcium channels in intestinal smooth muscles can be now added to the muscle relaxant effects of SchisQndra lignans.

In addition to the influx of extracellular [Ca.sup.2+], the release of intracellular [Ca.sup.2+] also plays an important role in smooth muscle contraction. Agonists (noradrenaline, acetylcholine, etc.) bind to the corresponding membrane receptors, and activate phospho-lipase C (PLC), which catalyzes the formation of two secondary messengers: inositol trisphosphate (IP3) and diacylglycerol (DG). The binding of IP3 to receptors on the sarcoplamic reticulum (SR) causes the release of [Ca.sup.2+] into cytosol, while DG activates protein kinase C (PKC) which phosphorylates specific target proteins, thereby sensitizing the contractile elements to [Ca.sup.2+] (Harnett and Biancani 2003; Webb 2003; Gerthoffer 2005). The present study showed that the contractions elicited by ACh in [Ca.sup.2+]-free solution were inhibited by schisandrin A in a concentration-dependent manner, which indicated that schisandrin A was able to inhibit the mobilization of intracellular [Ca.sup.2+] release activated by ACh, resulting in smooth muscle relaxation. In a previous study, magnolol, a major compound isolated from Cortex Magnolia officinalis, was also claimed to inhibit the [Ca.sup.2+] release from SR, which was a part of the mechanism(s) of its relaxant effect on guinea pig distal colon (Bian et al. 2006). The results in our study, however, have not identified a specific site for the inhibitory action of schisandrin A, for example, the phospholipase C or (and) IP3 receptors activation. Further work is needed to clarify the mechanism(s) of immobilizing intracellular [Ca.sup.2+] by schisandrin A.

In conclusion, the present study investigated the relaxant effects of Schisandra chinensis on smooth muscles of guinea pig ileum. The results demonstrate that the extracts of S. chinensis fruits caused relaxation on the contractile responses induced by ACh and 5-HT. The major lignan compounds, schisandrin, schisandrol B, schisandrin A and schisandrin B, also produced various inhibitory effects, and could be identified as the major active ingredients responsible for the antispasmodic activity. The relaxant action of schisandrin A involved inhibitions on influx of extracellular [Ca.sup.2+] and mobilization of intracellular [Ca.sup.2+] rather than antagonism of cholinergic M3 receptor. This study would suggest a role of S. chinensis in treating gastrointestinal diseases and the significance of lignans in this medicinal use.

Acknowledgments

The present study was partially supported by grant 1-U19-AT003266 (PI: Brian Berman, University of Maryland) from the National Center for Complementary and Alternative Medicine (NCCAM), NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCCAM. All members of this international collaborative project are acknowledged for their collaborative efforts. The authors are also grateful to Mr. Kam Ming Chan for his technical assistance in the tissue isolation experiments. Dr.Jia Ming Yang received a postgraduate studentship from the Chinese University of Hong Kong during this study.

0944-7113/$ - see front matter [c] 2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2011.06.003

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Jia-Ming Yang (a), Paul Siu Po Ip (a), Chun-Tao Che (a), (c), *, (1), John H.K. Yeung (b), **, (1),

(a) School of Chinese Medicine, Faculty of Science. The Chinese University of Hong Kong, Shatin Hong Kong

(b) School of Biomedical Sciences, Faculty of Medicine, Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

(c) Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60612, USA

* Corresponding author at: School of Chinese Medicine, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Tel.: +852 26098130; fax: +852 26037203.

** Corresponding author. Tel.: +852 26096864; fax: +852 26035139. E-mail addresses: chect@uic.edu (C.-T. Che), johnyeung@cuhk.edu.hk (J.H.K. Yeung).

(1) Both the authors contributed equally.
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Author:Yang, Jia-Ming; Ip, Paul Siu Po; Che, Chun-Tao; Yeung, John H.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Geographic Code:9HONG
Date:Oct 15, 2011
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