Hexane extract of Poncirus trifoliata (L.) Raf. stimulates the motility of rat distal colon

Hexane extract of Poncirus trifoliata (L.) Raf. stimulates the motility of rat distal colon

Journal of Ethnopharmacology 127 (2010) 718–724 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevie...

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Journal of Ethnopharmacology 127 (2010) 718–724

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm

Hexane extract of Poncirus trifoliata (L.) Raf. stimulates the motility of rat distal colon Keun Han Choi a,1 , Seung Il Jeong b,1 , Byung Soon Hwang b , Jun Ho Lee a , Hyun Kwang Ryoo a , Seoul Lee a , Bong Kyu Choi a , Kyu Yong Jung a,∗ a b

Department of Pharmacology, College of Medicine, Wonkwang University, 344-2 Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea Jeonju Biomaterials Institute, Jeonbuk 561-360, Republic of Korea

a r t i c l e

i n f o

Article history: Received 6 July 2009 Received in revised form 12 November 2009 Accepted 30 November 2009 Available online 4 December 2009 Keywords: Poncirus trifoliata (L.) Raf. Hexane extract Distal colon Motility Rat

a b s t r a c t Aim of the study: Poncirus trifoliata (L.) Raf. (Rutaceae, PT) has been commonly used for treating gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of colonic motility have not been clarified. This study investigated the regulatory effects of PT on the colonic motility. Materials and methods: Immature fruits of PT were sequentially partitioned with MeOH, n-hexane, CHCl3 , EtOAc, n-BuOH and H2 O, and the effects of PT extracts on the contractility of colonic strips and colonic luminal transit in rats were measured in vitro and in vivo, respectively. Results: Among six different extracts, only hexane extract of PT (PTHE) dose-dependently increased the low frequency contraction of longitudinal muscle in distal colonic strips, and the ED50 value was revealed to be 0.71 ␮g/ml. The contractile patterns induced by PTHE were remarkably different from those caused by acetylcholine (ACh) and serotonin (5-HT). The stimulatory effects of PTHE on the whole distal colonic strips were more prominent than on the mucosa/submucosa-denuded segments. The M2 receptor-preferring, methoctramine (0.5 ␮M), and M3 receptor-preferring antagonist, 4-DAMP (0.5 ␮M) significantly blocked the PTHE (1 ␮g/ml)-induced contraction of distal colon longitudinal muscles, whereas the 5-HT receptor antagonists (1.0 ␮M, alone or in combination) selective for 5-HT3 (ondansetron), 5-HT4 (GR113808) and 5-HT1, 2, 5–7 (methysergide) receptors did not change the PTHE (1 ␮g/ml)-induced contractility of distal colon longitudinal muscles. SNAP (0.1 mM), a NO donor, enhanced the stimulatory effects of PTHE on the longitudinal muscle of distal colon, but l-NAME (0.1 mM), a NO synthesis inhibitor, had no effects. PTHE (10–100 mg/kg) caused a dose-dependent increase of colonic luminal transit. Conclusions: Collectively, these findings suggest that PTHE specifically acts on the longitudinal muscle of distal colon in rats, and these stimulatory effects are likely mediated, at least, by activation of acetylcholinergic M2 and M3 receptors. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The immature fruits of Poncirus trifoliata (L.) Raf. (Rutaceae, PT) have been known to reveal a variety of pharmacological properties such as anti-oxidant (Jayaprakasha et al., 2007), anti-platelet (Teng et al., 1992), anti-bacterial (Kim et al., 1999a) and antiallergic (Lee et al., 1996) activities and to contain more than 50 phytochemicals including poncirin, limonene, synephrine, hesperidin, neohesperidin, auraptene and imperatorin (Kim et al., 1999b, 1997).

∗ Corresponding author. Tel.: +82 63 850 6796; fax: +82 63 851 0879. E-mail address: [email protected] (K.Y. Jung). 1 These authors equally contributed to this work. 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.11.032

Decoction of PT is commonly used for treating and/or alleviating the symptoms of gastrointestinal (GI) disorders related with abnormal GI motility and gastric secretion in Korean traditional medicine (Kim et al., 1997). Despite the considerable use of PT for GI dysfunction, as far as we know, pharmacological roles of PT in the regulation of colonic motility have not been understood. It has been experimentally demonstrated that PT accelerates the small bowel transit of rats, whereas it has no effects on the gastric emptying (Lee et al., 2005a). The therapeutic property of PT for treating abnormal GI motility has been demonstrated in vivo animal study (Lee et al., 2005b), indicating that PT stimulates small bowel transit in ileus-induced rats and morphine-treated mice. These results obviously support the therapeutic potential of PT as a remedy for treatment of GI motility disorders. However, the stimulatory effects of PT on the ileum contractility alone may be not enough to understand the improvement of

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propulsive GI motility for defecation in vivo, because colonic contractility is generally though of as providing the major mechanical force behind intestinal propulsion (Cellek et al., 2006; Gonzalez and Sarna, 2001). In view of functional importance of colonic motility for migrating GI contents into rectum and therapeutic use of PT to improve the defecation disorders in Korean traditional medicine, we have hypothesized that PT possibly increases the contractility of colonic smooth muscles, results in facilitating the physiological function of defecation. In order to examine these hypotheses, using pharmacological strategies, this study investigated the regulatory effects of PT extracts in the spontaneous contractility of colonic smooth muscle strips prepared from rats and in the facilitation of rat colonic transit in vitro and in vivo, respectively.

and pinned mucosa side up to the base of a Sylgard-coated dish contained with Krebs–Ringer bicarbonate buffer. The mucosa and submucosa were carefully dissected away using small forcep and ophthalmological trabecula scissor, and the isolated muscle strips (1.5 cm × 1 cm) were prepared. The whole or isolated strips were longitudinally or circularly mounted in an organ bath (10 ml) containing Krebs–Ringer bicarbonate buffer bubbled with 5% CO2 /95% O2 and maintained at 37 ◦ C. One edge of colonic strips tied with suture silk was fixed to the bottom of organ bath, and the other edge was connected to the isometric force transducer (Grass Technologies, West Warwick, RI).

2. Materials and methods

The contractile responses of colonic strips to PT extracts were measured as described previously (Gonzalez and Sarna, 2001; Jeong et al., 2009). In brief, after strips were allowed to equilibrate for 60–90 min with washout every 10 min, 1 g of tension was slowly applied to the tissue before treating the drugs. The strips were washed at least 3 times (10–15 min intervals) with Krebs–Ringer bicarbonate buffer between each experimental condition. All antagonists used were pretreated for 5 min before treating agonists including PT extracts. Acetylcholine (ACh) and serotonin (5-HT) were used as positive control drugs in comparison with the PT extract-induced colonic contractility. The mean amplitude of colonic contraction produced under the resting and drug-treated conditions was measured over period of 10 min and 15–20 min, respectively. Contractility outputted from the force transducer was isometrically measured by biological recording system equipped with amplifier (PowerLab 4/25, AD Instruments, Colorado Spring, CO). The ED50 values were interpolated from concentration–response curve by means of personal computer program (GraphPad Prism and Instat 5.5).

2.1. Plant material The immature fruits of PT were collected at Jinan, Jeonbuk, Korea in May 2008, and authenticated by Prof. Young Sung Ju, College of Oriental Medicine, Woosuk University (Jeonju, Korea). A voucher specimen (JSI119) was deposited in the co-author’s laboratory, Jeonju Biomaterial Institute. 2.2. Poncirus trifoliata extracts The dried and grounded immature fruits of PT (600 g) were refluxed with MeOH for 4 h (3× 6 l). The total filtrates were then concentrated to dryness in vacuo at 40 ◦ C to give a MeOH extract (72 g). These extracts were suspended in H2 O and then successively partitioned with n-hexane, CHCl3 , EtOAc and n-BuOH to give nhexane (6.7 g), CHCl3 (8.9 g), EtOAc (5.6 g) and n-BuOH (19.2 g), as well as H2 O (28.4 g) residues. All extracts, except H2 O fraction, were dissolved with 10% dimethyl sulfoxide (DMSO) to be used in the experiments, and the final concentration of DMSO was less than 0.1%, which did not affect the spontaneous contractility of colonic smooth muscles (data not shown). 2.3. Animals This study used 8-week-old male Sprague–Dawley rats received from the Samtako BioKorea (Kyungki, Korea), and all animals were acclimated for 1 week with free access to a solid rodent diet (Samyang Co., Kyungki, Korea) and tap water under controlled conditions of a temperature 23 ± 2 ◦ C, relative humidity 50–60% and 12 h dark–light cycle. Animals were deprived of food for 20 h before experiments, but they were allowed free access to tap water. All experiments were conducted in accordance with the institutional guideline established by the Wonkwang University Committee for the Care and Use of Laboratory Animals. 2.4. Whole and isolated tissue preparation Animals were sacrificed by CO2 asphyxiation and cervical dislocation, and two different types of colonic strips, whole and isolated strip, were prepared. Whole strips were obtained by excising the entire tissue of proximal and distal colons, and the isolated smooth muscle strips were prepared by removing the mucosa and submucosa of distal colon. For whole tissue preparation, strips (1.5 cm) of proximal colon in the distance of 1 cm from the ileo-cecal junction and distal colon in the distance of 1 cm from the anus were dissected, and luminal contents were flushed out using Krebs–Ringer bicarbonate buffer (composition 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , 2.6 mM CaCl2 , 25 mM NaHCO3 and 11.5 mM d-glucose, pH 7.4). For preparing the isolated strips, distal colon was opened along the mesenteric border

2.5. Measurement of colonic contractility

2.6. Construction of concentration–response curves Concentration–response curves, increasing the concentration of hexane extract of PT (PTHE, 0.05–5 ␮g/ml), were applied using a “single-dose” protocol. The longitudinally or circularly mounted whole and isolated distal colons were treated with each concentration of PTHE for 15 min, followed by washing 5 times with fresh Krebs–Ringer bicarbonate buffer. Each strip was used for only one discrete concentration–response curve. The time interval between successive PTHE applications varied from 40 to 50 min, since more times were required to recover spontaneous activity after administration of higher concentration of PTHE. 2.7. Measurement of colonic transit Colonic luminal transit in rats was investigated with a slight modification of the previously reported method (Nagakura et al., 1997; Tsubouchi et al., 2003). In brief, rats were anesthetized with ketamine (30 mg/kg, i.p.), and the cecum was exposed by abdominal middle incision. An indwelling polyethylene cannula (I.D., 0.58 mm; O.D., 0.97 mm; PE-50, Becton Dickinson and Co., Parsippany, NJ) was implanted into the proximal colon about 1.5 cm from the cecum, and the other end of cannula was led to the interscapular region of the animal’s neck. The abdominal middle incision was closed with suture. Rats were individually housed and allowed to recover from surgery for 5 days. Rats were deprived of food for 20 h before colonic transit study, and all experiments were performed under conscious and freely moving condition. Immediately before administration, PTHE and itopride were dissolved in 0.5% carboxymethyl cellulose and saline, respectively. PTHE (10–100 mg/kg) and itopride (10 mg/kg) were orally administered at 30 min before gentle infusion of marker

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[barium sulfate, 60% (w/v), 0.5 ml/animal] into the proximal colon through the implanted cannula. Atropine (50 mg/kg) was subcutaneously injected at 5 min before administration of PTHE. Animals were sacrificed by cervical dislocation at 60 min after administration of marker. Entire colon was carefully and quickly removed, and the length from the colo-cecal junction to the front traveling edge of the marker was measured. Colonic luminal transit was expressed as the percentage of distance traversed to the total length of colon.

2.8. Chemicals and reagents ACh, atropine, 4-diphenyllacetoxy-N(2-chloriethyl)-piperidine (4-DAMP), methoctramine, 5-HT, ondansetron, GR113808, methysergide, S-nitroso-N-acetyl-penicillamine (SNAP), Nw nitro-l-arginine methyl ester (l-NAME) and barium sulfate were purchased from the Sigma Chemicals (St. Louis, MO). Itopride hydrochloride was obtained from the Abbott Japan Co. (Katsuyama, Japan). All other chemicals and reagents were of the highest grade from commercial sources.

2.9. Statistical analysis All results are presented as means ± S.E. of 7–9 experiments. Statistical analysis of the in vivo data was performed with William’s multiple range test. For the in vitro results, one-way analysis of variance (ANOVA) was performed, followed by post hoc analysis by Student–Newman–Keuls test. Probability values less than 0.05 were considered statistically significant.

3. Results 3.1. Effect of PT extracts on colonic contractility The components of PT were sequentially fractionated by organic system (MeOH, n-hexane, CHCl3 , EtOAc, n-BuOH and H2 O), and the effects of each extract on the colonic contractility were examined. Among six fractionations, only PTHE significantly stimulated the magnitude of low frequency contraction in the longitudinal muscle of distal colon (Fig. 1), whereas it did not change the spontaneous contractility of proximal colon longitudinal muscle (Fig. 1A). The other fractions, except PTHE, did not affect the spontaneous contractility of proximal and distal colon longitudinal muscle (representative tracings not shown). Fig. 1B shows the extracts-induced mean magnitude of low frequency contraction in the proximal and distal colon longitudinal muscle.

3.2. Comparison of contractile pattern PTHE reproduced the typical pattern of longitudinal muscle contractility in distal colon (Fig. 2A): a significant increase (magnitude and frequency) of low frequency contraction with high amplitude was recorded from the distal colonic strips after treatment of PTHE (1.0 ␮g/ml), and the stimulatory response was observed up to 2 h tested here. However, PTHE did not significantly affect the high frequency contraction with low amplitude. These patterns in time course and magnitude markedly differed from those produced by ACh (0.1 ␮M) and KCl (60 mM), which caused a tonic contraction immediately after treatment of agonists, followed by a gradual decline to the resting levels (Fig. 2). Comparing the whole distal colon, stimulatory effects of PTHE on the low frequency contraction were markedly attenuated in the isolated strips (Fig. 2B). There was no significant difference of ACh- or high KCl-induced contractile pattern between the whole and isolated distal colons.

Fig. 1. Effects of Poncirus trifoliata extracts on the spontaneous contractility of colonic longitudinal muscle. The whole (intact) proximal and distal colonic strips were longitudinally mounted in an organ bath, and contractile responses of strips to the extracts were measured by isometric force transducer before (10 min) and after (15 min) treatment of extracts. (A) Representative tracings produced by hexane extract. (B) The percentage change of contractility. Results are expressed as mean ± S.E. of 7–9 preparations. **P < 0.01 compared to the corresponding values of basal contractility.

3.3. Dose–response of hexane extract The stimulatory effects of PTHE on the contractility of distal colon according to the orientation of smooth muscle were examined using the longitudinal and circular muscle of whole and isolated distal colon. PTHE at 1 ␮g/ml significantly stimulated the low frequency contraction only in the longitudinal muscle with whole tissues, but not in the other strips (Fig. 3A). Additionally, PTHE (0.05–5 ␮g/ml) dose-dependently stimulated the magnitude of low frequency contraction in longitudinal muscle of whole distal colon, whereas it up to 5 ␮g/ml did not change them in the others (Fig. 3B). The ED50 value in stimulating the low frequency contraction in the longitudinal muscle of whole distal colon was estimated to be 0.71 ␮g/ml. 3.4. Antagonistic effect on the PTHE-induced contraction The cellular pathways implicated in the PTHE-stimulated longitudinal muscle contraction of whole distal colonic strips were examined using pharmacological strategy. The muscarinic receptor antagonists (0.5 ␮M) selective for the muscarinic (atropine), M2 (methoctramine) and M3 (4-DAMP) receptors significantly reduced the magnitude of ACh (0.1 ␮M)-induced contraction, and similar inhibitory effects were also observed in the PTHE (1 ␮g/ml)induced contraction of distal colon longitudinal muscle (Fig. 4A). Methysergide (1 ␮M) selective for 5-HT1, 2, 5–7 receptors significantly inhibited the 5-HT (10 ␮M)-induced contractility of distal colon longitudinal muscle, but no effects on the PTHE (1.0 ␮g/ml)-

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Fig. 2. Contractile patterns produced by the hexane extract of Poncirus trifoliata in the longitudinal muscle of distal colon. The whole (intact) and isolated (mucosa and submucosa denuded) distal colonic strips were longitudinally mounted in an organ bath, and the contractile response to stimulators was recorded. (A) Representative tracings produced by hexane extract or ACh in whole distal colonic strips. (B) Typical tracings in the whole and isolated distal colonic strips.

induced contraction (Fig. 4B). In the presence of methysergide, 5-HT antagonists (1.0 ␮M, alone or in combination) selective for 5HT3 (ondansetron) and 5-HT4 (GR113808) receptors significantly reduced the magnitude of the 5-HT-induced contractility of distal colon longitudinal muscle, but not the contraction stimulated by PTHE (Fig. 4B). 3.5. Nitric oxide on the PTHE-induced contraction This study also examined whether PTHE might regulate the nitric oxide (NO) pathways in the longitudinal muscle of distal colon. As shown in Figs. 5A and B, pretreatment of whole distal colonic strips with NO donor, SNAP (0.1 mM), significantly enhanced the PTHE (1 ␮g/ml)-induced low frequency contraction, whereas inhibition of NO synthesis by l-NAME (0.1 mM) had no significant effect on the colonic contractility stimulated by PTHE. 3.6. Colonic luminal transit The colonic luminal transit was studied to examine the effects of PTHE on colonic motility in vivo, and the obtained results are shown in Fig. 6. Itopride (10 mg/kg) to be used as a positive control drug significantly accelerated the colonic transit of rats. Oral administration of PTHE-stimulated the colonic luminal transit in a dose-dependent manner (10–100 mg/kg), and PTHE (100 mg/kg)-induced colonic luminal transit was significantly attenuated by preadministration of atropine (50 mg/kg). 4. Discussion This study provides the spasmogenic effects of PTHE on the distal colon longitudinal muscle in relation to the cholinomimetic

Fig. 3. Effects of hexane extract of Poncirus trifoliata on the spontaneous contractility of longitudinal and circular muscle in the whole (intact) and isolated (mucosa and submucosa denuded) distal colon. The strips were longitudinally or circularly mounted in an organ bath, and the contractile response to hexane extract was measured by isometric force transducer before (10 min) and after (15 min) treatment of extracts. (A) Representative tracings. (B) The percentage changes of contractility. Results are expressed as mean ± S.E. of 7 preparations. **P < 0.01 compared to the corresponding values of basal contractility.

mechanism both in vitro and in vivo. Among six different organic extracts fractionated here, only hexane extract, PTHE, produced a significant contractile response to the longitudinal muscle of distal colon. PTHE caused a dose-dependent increase in the low frequency contraction of distal colon longitudinal muscle, and these stimulatory effects were likely mediated via the M2 and M3 receptors as judged by the sensitivity of contractile response to blockade by the M2 - and M3 -selective antagonists. PTHE also significantly accelerated the colonic luminal transit in vivo. Therefore, the findings observed here possibly support the common use of PT to treat and/or ameliorate the GI disorders related with abnormal GI motility in Korean traditional medicine. Although PT is commonly used as a remedy to improve the dysfunction of GI motility and secretion in Korean traditional medicine (Kim et al., 1997), the pharmacological roles of PT in the regulation of colonic motility have not been clarified yet. Recently, Lee et al. (2005a,b) have reported the prokinetic activity of PT, suggesting

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Fig. 4. Effects of neurotransmitter receptor antagonists on the contraction of distal colon longitudinal muscle stimulated by the hexane extract of Poncirus trifoliata. After whole (intact) distal colonic strips were longitudinally mounted in an organ bath, the muscarinic (A) or 5-HT (B) receptor antagonists were treated for 5 min, followed by treatment of agonists. The contractility was measured by isometric force transducer before (10 min) and after (20 min) treatment of agonists. Results are expressed as mean ± S.E. of 7 preparations. *P < 0.05 and **P < 0.01 compared to control.

that aqueous extracts of PT accelerate small bowel transit in normal and ileus-induced rats, but no effects on the gastric emptying. Using six different organic systems, we partially purified the phytochemical components contained in PT and observed that only non-polar n-hexane fraction of PT, PTHE, significantly stimulated the low frequency contraction of distal colon. Moreover, the stimulatory effects of PTHE were observed only in the longitudinal muscle of distal colon, not in the circular muscle of distal colon and in the longitudinal and circular muscle of proximal colon. These findings suggest that the main effective ingredients of PT to stimulate the distal colonic contractility are likely contained in the n-hexane fraction, and PTHE likely has a region (proximal and distal)- and orientation (longitudinal and circular)-specific activity in the stimulation of low frequency contraction of distal colon longitudinal muscle. Comparing the spontaneous contractility of gastric and small intestinal smooth muscle, the colonic smooth muscle has a unique contractile pattern consisting of two different types (low and high frequency) of contraction, and their amplitude and frequency vary according to the region (proximal and distal) of colon and orientation (longitudinal and circular) of smooth muscle (Alberti et al., 2005). These unique contractile patterns of colonic smooth muscle were also observed in this study, showing that colonic strips under resting condition apparently produced the spontaneous low (with high amplitude) and high (with low amplitude) frequency contraction, and the frequency and amplitude of low frequency contraction

Fig. 5. Effects of NO on the longitudinal muscle contraction stimulated by the hexane extract of Poncirus trifoliata in distal colon. After whole distal colonic strips were longitudinally mounted in an organ bath, SNAP or l-NAME was treated for 5 min, followed by treatment of hexane extract for 20 min. The contractility was measured by isometric force transducer before (10 min) and after (20 min) treatment of extract. (A) Representative tracings. (B) The percentage change of contractile response. Results are expressed as mean ± S.E. of 7 preparations. *P < 0.05 compared to control.

Fig. 6. Effects of hexane extract of Poncirus trifoliata on the colonic transit in rats. Itopride or hexane extract was orally administered at 30 min before administering marker, and atropine was subcutaneously injected at 5 min before administrating marker. Animals were sacrificed at 60 min after administration of marker. Each bar represents the mean ± S.E. of 6 rats. *P < 0.05 and **P < 0.01.

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were obviously different between proximal and distal colon or longitudinal and circular muscle. These results suggest that GI strips used in this study may be useful to evaluate the pharmacological property of PTHE in the regulation of colonic contractility. We also observed that ACh and 5-HT caused a tonic contraction immediately after treatment of agonists in the longitudinal muscle of distal colon, followed by a gradual decline to the resting level with a slight increase of low frequency contraction. In contrast, PTHE produced a sustained increase of low frequency contraction without tonic contraction. These apparent differences possibly reflect the unique pharmacological and/or therapeutical properties of PT in the regulation of GI disorders. As far as we know, the cellular pathways implicated in the PTinduced contraction of GI smooth muscle have not been understood yet. We are then interested in the evaluation of pharmacological mechanisms involved in the PTHE-induced contraction of distal colon longitudinal muscle. It has been widely known that cholinomimetic mechanism may be primarily involved in the excitatory action of GI motility, and the M2 and M3 receptors among five different subtypes of muscarinic receptors play a key role in the smooth muscle contraction of GI tract (Matsui et al., 2002; Takeuchi et al., 2005; Unno et al., 2005). Therefore, using muscarinic receptor-specific antagonists, we examined whether PTHE-stimulated the low frequency contraction of distal colon longitudinal muscle through activating the cholinomimetic pathway. All muscarinic receptor antagonists used significantly blocked the ACh- and PTHE-induced contraction of longitudinal muscle in distal colon. Interestingly, inhibitory ratio of methoctramine on the PTHE-induced contraction was much higher than that of the AChinduced contraction. These findings suggest that compared to ACh, PTHE may have high affinity with the M2 receptors, and PTHEinduced contraction of distal colon longitudinal muscle is possibly mediated, at least, by activation of the M2 and M3 receptors. These suggestions can be also supported by the results, showing that PTHE remarkably stimulated the colonic luminal transit in rats, and the enhanced colonic transit was significantly blocked by administration of atropine. To understand clearly the involvement of cholinomimetic pathway in the PTHE-induced distal colonic contraction, further studies such as binding assay are needed to elucidate the pharmacological affinity of PTHE with muscarinic receptor subtypes. Among seven different types of serotoninergic receptors, the 5-HT3 and 5-HT4 receptors are the most thoroughly understood subtypes with the regards to physiological function and histological distribution in GI tract (Chetty et al., 2006; Cellek et al., 2006). The 5-HT3 receptor induces a rapid depolarization of the myenteric neuron through enhancing ACh release (Kim and Camilleri, 2000; Talley, 2001), and 5-HT4 receptor expressed in the nerve terminal facilitates the releases of neurotransmitters including ACh, substance P and vasoactive intestinal peptides (Jin et al., 1999; Talley, 2001). These cellular events possibly lead us to consider that 5-HT acts as an up-stream regulator to enhance the excitatory activity of GI smooth muscles through mediating the ACh release. In this study, we observed that 5-HT stimulated the spontaneous contractility of longitudinal muscle in distal colon, and the stimulatory effects were significantly blocked by 5-HT receptor antagonists (alone or in combination). In contrast, the 5-HT antagonists had no effects on the PTHE-induced contraction of distal colon. Although it has been suggested that 5-HT can produce the different responses such as muscle contraction or relaxation depending on the experimental conditions (Kim and Camilleri, 2000), the obtained results suggest that serotoninergic pathway is not likely implicated in the PTHE-induced contraction of distal colon longitudinal muscle. The low frequency contraction with high amplitude in an organ bath study can be recorded and referred to as a giant contraction (Hoogerwerf, 2006; Gonzalez and Sarna, 2001). The low frequency

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contraction of distal colonic strips is primarily generated by nonadrenergic, non-cholinergic (NANC) NO pathways (Gonzalez and Sarna, 2001; Middleton et al., 1993). Interestingly, SNAP enhanced the PTHE-induced contraction of distal colon longitudinal muscle, but l-NAME did not affect. Although we do not presently have direct evidences to understand the cellular mechanisms underlying the stimulatory or inhibitory effects of endogenous NO modulators on the PTHE-induced contraction of colonic smooth muscle, we consider that NO donated by SNAP facilitates the cholinergic neurotransmission in ascending excitatory nervous pathway to both longitudinal and circular muscle of distal colon through soluble guanylate cyclase (Smith and McCarron, 1998), which produces the cGMP, resulted in activation of cGMP-dependent protein kinase to phosphorylate myosin and other proteins that regulate smooth muscle contraction (Mustafa et al., 2009). These signaling events possibly enhance the PTHE-induced muscle contraction of distal colon. In addition, it has been also known that NO suppresses the cholinergic neurotransmission in descending inhibitory nervous pathway (Smith and McCarron, 1998), and it enhances the colonic transit by mediating descending relaxation, which in turn, facilitates propulsion of the colonic contents (Mizuta et al., 1999). Therefore, we cannot rule out the possibility that stimulatory effects of NO donor (SNAP) on the PTHE-induced colonic muscle contraction are likely related with the experimental conditions. Anyhow, further studies are needed to elucidate the regulatory roles of NO in the PTHE-induced contraction colonic smooth muscle. In conclusion, the results found in this study suggest that hexane extract of PT specifically acts on the longitudinal muscle of distal colon in rats, and its stimulatory activity is likely mediated, at least, by activation of ACh receptors, both the M2 and M3 receptors. The present study provides information to understand the therapeutic property of PT for modulating the GI motility in Korean traditional medicine. Acknowledgement This study was supported by Wonkwang University Research Grant in 2008. References Alberti, E., Mikkelsen, H.B., Larsen, J.O., Jimenez, M., 2005. Motility patterns and distribution of interstitial cells of Cajal and nitrergic neurons in the proximal, midand distal-colon of the rat. Neurogastroenterology and Motility 17, 133–147. Chetty, N., Irving, H.R., Coupar, I.M., 2006. Activation of 5-HT3 receptors in the rat and mouse intestinal tract: a comparative study. British Journal of Pharmacology 148, 1012–1021. Cellek, S., John, A.K., Thangiah, R., Dass, N.B., Bassil, A.K., Jarvie, E.M., Lalude, O., Vivekanandan, S., Sanger, G.J., 2006. 5-HT4 receptor agonists enhance both cholinergic and nitrergic activities in human isolated colon circular muscle. Neurogastroenterology and Motility 18, 853–861. Gonzalez, A., Sarna, S.K., 2001. Neural regulation of in vitro giant contractions in the rat colon. American Journal of Physiology 281, G275–G282. Hoogerwerf, W.A., 2006. Prokineticin 1 inhibits spontaneous giant contractions in the murine proximal colon through nitric oxide release. Neurogastroenterology and Motility 18, 455–463. Jin, J.G., Foxx-Orenstein, A.E., Grider, J.R., 1999. Propulsion in guinea pig colon induced by 5-hydroxytrytamine (5-HT) via 5-HT4 and 5-HT3 receptors. Journal of Pharmacology and Experimental Therapeutics 288, 93–97. Jeong, S.I., Kim, Y.S., Lee, M.Y., Kang, J.K., Lee, S., Choi, B.K., Jung, K.Y., 2009. Regulation of contractile activity by magnolol in the rat isolated gastrointestinal tracts. Pharmacological Research 59, 183–188. Jayaprakasha, G.K., Mandadi, K.K., Poulose, S.M., Jadegoud, Y., Nagana Gowda, G.A., Patil, B.S., 2007. Inhibition of colon cancer cell growth and antioxidant activity of bioactive compounds from Poncirus trifoliata (L.) Raf. Bioorganic and Medicinal Chemistry 15, 4923–4932. Kim, D.Y., Camilleri, M., 2000. Serotonin: a mediator of the brain-gut connection. American Journal of Gastroenterology 95, 2698–2709. Kim, D.H., Bae, E.A., Han, M.J., 1999a. Anti-helicobacter pylori activity of the metabolites of poncirin from Poncirus trifoliata by human intestinal bacteria. Biological and Pharmaceutical Bulletin 22, 422–424.

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Kim, H.M., Kim, H.J., Park, S.T., 1999b. Inhibition of immunoglobulin E production by Poncirus trifoliata fruit extract. Journal of Ethnopharmacology 66, 283–288. Kim, C.M., Shin, M.K., Ahn, D.G., Lee, K.S., 1997. Chungyak Daesajun, vol. 8. Jungdam Publisher, Seoul, pp. 3969–3976. Lee, Y.M., Kim, D.K., Kim, S.H., Shin, T.Y., Kim, H.M., 1996. Antianaphylactic activity of Poncirus trifoliata fruit extract. Journal of Ethnopharmacology 54, 77–84. Lee, H.T., Seo, E.K., Chung, S.J., Shim, C.K., 2005a. Prokinetic activity of an aqueous extract from dried immature fruit Poncirus trifoliata (L.) Raf. Journal of Ethnopharmacology 102, 131–136. Lee, H.T., Seo, E.K., Chung, S.J., Shim, C.K., 2005b. Effect of an aqueous extract of dried immature fruit of Poncirus trifoliata (L.) Raf. on intestinal transit in rodents with experimental gastrointestinal motility dysfunctions. Journal of Ethnopharmacology 102, 302–306. Middleton, S.J., Cuthbert, A.W., Shorthouse, M., Hunter, J.O., 1993. Nitric oxide affects mammalian distal colonic smooth muscle by tonic neural inhibition. British Journal of Pharmacology 108, 974–979. Mustafa, A.K., Gadalla, M.M., Snyder, S.H., 2009. Signaling by gasotransmitters. Science Signaling 2, re2. Matsui, M., Motomura, D., Fujikawa, T., Jiang, J., Takahashi, S., Manabe, T., Taketo, M.M., 2002. Mice lacking M2 and M3 muscarinic acetylcholine receptors are devoid of cholinergic smooth muscle contractions but still viable. The Journal of Neuroscience 22, 10627–10632. Mizuta, Y., Takahashi, T., Owyang, C., 1999. Nitrergic regulation of colonic transit in rats. American Journal of Physiology 277, G275–G279.

Nagakura, Y., Kontoh, A., Tokita, K., Tomoi, M., Shimomura, K., Kadowaki, M., 1997. Combined blockade of 5-HT3 - and 5-HT4 -serotonin receptors inhibits colonic functions in conscious rats and mice. Journal of Pharmacology and Experimental Therapeutics 281, 284–290. Smith, T.K., McCarron, S.L., 1998. Nitric oxide modulates cholinergic reflex pathways to the longitudinal and circular muscle in the isolated guinea-pig distal colon. Journal of Physiology 512, 893–906. Talley, N.J., 2001. Serotoninergic neuroenteric modulators. Lancet 358, 2061–2068. Takeuchi, T., Fujinami, K., Goto, H., Fujita, A., Taketo, M.M., Manabe, T., Matsui, M., Hata, F., 2005. Roles of M2 and M4 muscarinic receptors in regulating acetylcholine release from myenteric neurons of mouse ileum. Journal of Neurophysiology 93, 2841–2848. Teng, C.M., Li, H.L., Wu, T.S., Huang, S.C., Huang, T.F., 1992. Antiplatelet actions of some coumarin compounds isolated from plant sources. Thrombosis Research 66, 549–557. Tsubouchi, T., Saito, T., Mizutani, F., Yamauchi, T., Iwanaga, Y., 2003. Stimulatory action of itopride hydrochloride on colonic motor activity in vitro and in vivo. Journal of Pharmacology and Experimental Therapeutics 306, 787–793. Unno, T., Matsuyama, H., Sakamoto, T., Uchiyama, M., Izumi, Y., Okamoto, H., Yamada, M., Wess, J., Komori, S., 2005. M2 and M3 muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice. British Journal of Pharmacology 146, 98–108.