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Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Gastrointestinal effect of methanol extract of Radix Aucklandiae and selected active substances on the transit activity of rat isolated intestinal strips Huimin Guo, Jingze Zhang, Wenyuan Gao, Zhuo Qu & Changxiao Liu To cite this article: Huimin Guo, Jingze Zhang, Wenyuan Gao, Zhuo Qu & Changxiao Liu (2014) Gastrointestinal effect of methanol extract of Radix Aucklandiae and selected active substances on the transit activity of rat isolated intestinal strips, Pharmaceutical Biology, 52:9, 1141-1149, DOI: 10.3109/13880209.2013.879601 To link to this article: http://dx.doi.org/10.3109/13880209.2013.879601

Published online: 20 Mar 2014.

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Date: 17 January 2017, At: 18:00

http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(9): 1141–1149 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.879601

ORIGINAL ARTICLE

Gastrointestinal effect of methanol extract of Radix Aucklandiae and selected active substances on the transit activity of rat isolated intestinal strips Huimin Guo1*, Jingze Zhang2*, Wenyuan Gao1, Zhuo Qu1, and Changxiao Liu3 1

Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China, 2Department of Pharmacy, Logistics College of Chinese People’s Armed Police Forces, Tianjin, China, and 3The State Key Laboratories of Pharmacodynamics and Pharmacokinetics, Tianjin, China Abstract

Keywords

Context: Radix Aucklandiae, the dry rhizome of Aucklandia lappa Decne (Asteraceae), enjoyed traditional popularity for its antidiarrheal effects. Although there are many investigations on its chemical constituents and pharmacologic actions, few studies explaining its activity and mechanism in gastrointestinal disorders are available. Objective: In this paper, we focused on the effects of the methanol extract of R. Aucklandiae (RA ext) on gastrointestinal tract, so as to assess some of the possible mechanisms involved in the clinical treatment. Materials and methods: In vivo, in neostigmine-induced mice and normal mice, after intragastric administration, RA ext (100, 200, 300, and 400 mg/kg) was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effect of it (0.1, 0.2, 0.3, and 0.4 mg/mL) on the isolated tissue preparations of rat jejunum was also investigated, as well as costunolide and dehydrocostuslactone which were the main constituents. Results: In vivo, the gastric emptying increased and intestinal transit decreased after the administration of RA ext in normal mice. However, RA ext inhibited the gastric emptying and the intestinal transit throughout the concentrations in neostigmine-induced mice. In vitro, RA ext caused inhibitory effect on the spontaneous contraction of rat-isolated jejunum in a dose-dependent manner ranging from 0.1 to 0.4 mg/mL, and it also relaxed the acetylcholine chloride (Ach, 105 M), 5-hydroxytryptamine (5-HT, 200 mM)-induced, and K+ (60 mM)-induced contractions. RA ext shifted the Ca2+ concentration–response curves to right, similar to that caused by verapamil (0.025 mM). The Ca2+ concentration–response curves were shifted by costunolide (CO) (5.4, 8.1, and 10.8 mg/mL), dehydrocostuslactone (DE) (4.6, 6.9, and 9.2 mg/mL), costunolide–dehydrocostuslactone (CO–DE) (5.4–4.6, 8.1–6.9, and 10.8–9.2 mg/mL) to the right, similar to that caused by verapamil (0.01 mM). Discussion and conclusion: These results indicate that RA ext played a spasmolytic role in gastrointestinal motility, which is probably mediated through the inhibition of muscarinic receptors, 5-HT receptors, and calcium influx. The presence of cholinergic and calcium antagonist constituents may be the compatibility of CO and DE. All these results provide a pharmacological basis for its clinical use in the gastrointestinal tract.

Cholinergic and calcium antagonist, gastric emptying, intestinal smooth muscle, small intestinal motility

Introduction According to the World Health Organization, there are approximately 2 billion annual cases of diarrhea worldwide. Diarrhea is the leading cause of death in children younger than 5 years old and kills 1.5 million children each year. It is especially prevalent in the developing world, where

*These two authors contributed equally to this work. Correspondence: Wenyuan Gao, School of Pharmaceutical Science and Technology, Tianjin University, Weijin Road, Tianjin 300072, China. Tel: +86 22 87401895. Fax: +86 22 87401895. E-mail: biochemgao@ 163.com

History Received 6 October 2013 Revised 28 November 2013 Accepted 12 December 2013 Published online 20 March 2014

mortality is related to dehydration, electrolyte disturbance, and the resultant acidosis. Additionally, diarrhea is also a common problem in the developed world, with 211–375 million episodes of infectious diarrheal illnesses in the United States annually resulting in 73 million physician consultations, 1.8 million hospitalizations, and 3100 deaths. Furthermore, 4–5% of the Western population suffer from chronic diarrhea (Kent & Banks, 2010). Given the high prevalence of diarrhea, this research has been directed at learning more about effective drugs in treating diarrhea. In recent years, as traditional Chinese medicine (TCM) plays an increasingly important role in our daily life, many patients turn to TCM for possible cure of diarrhea.

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O

O

O Costunolide

Dehydrocostuslactone

Figure 1. Chemical structures of costunolide and dehydrocostuslactone.

Radix Aucklandiae (RA), the dry rhizome of Aucklandia lappa Decne, commonly known as Muxiang in China, has been recorded in the Chinese Pharmacopoeia (Committee of National Pharmacopoeia, 2010). As a traditional Chinese medicine, it was always suggested in prescriptions or used as an herb for the treatment of gastrointestinal dysfunction. It not only has anti-inflammatory (Gokhale et al., 2002) and antitumor (Jung et al., 1998) effects but can also improve the symptoms of asthma, cough, diarrhea, vomiting, indigestion, colic, cholecystitis, and hepatitis (Yamahara et al., 1985). Phytochemical investigations have shown that sesquiterpene lactones were the effective components in this herb. Among them, the major compositions are costunolide (CO) and dehydrocostuslactone (DE), whose chemical structures are shown in Figure 1. CO and DE have a variety of pharmacological activities. On one hand, they have effects of antibacterial, antalgic, and antivirus; on the other hand, they could dilate bronchus, improve stomach function, depress blood pressure, and relieve the spasm of smooth muscle (Aifeng et al., 2005). Consequently, the content of CO and DE has been used as the standard index to evaluate the quality of RA. Numerous studies have validated the traditional use of antidiarrheal medicinal plants by investigating the biological activity of such plants, which have antispasmodic effects, could delay intestinal transit, suppress gut motility, stimulate water adsorption, or reduce electrolyte secretion (Aifeng et al., 2005; Brunton, 1996; Godfraind et al., 1986; Palombo, 2006). In this study, the effects of the RA ext on gastric emptying and small intestinal transit of mice in vivo, and on the smooth muscle contractions in vitro using the isolated rat jejunum were investigated. In order to understand the mechanism, we also investigated the effects of the two main active chemical components on isolated smooth muscle of gastrointestinal tract and their relationship with Ca2+.

Materials and methods Plant material and preparation of Radix Aucklandiae RA was provided by Tianjin Lerentang Pharmaceutical Factory (Tianjin, China) and identified by Professor Wenyuan Gao from School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China. The voucher specimens (Voucher no. MX100108) are available in the herbarium of Research Center of Tianjin Zhongxin Pharmaceuticals, Tianjin, China. All the reagents were of analytical grade. RA (200 g) was powdered and extracted with 2000 mL of methanol for 1 h, three times, and the extract

obtained was evaporated under reduced pressure to remove methanol and concentrated to obtain the crude extract (MeOH extract, 62.6 g) at a temperature below 50 C. The yield was 31.3%. The RA ext was grinded with Tween 80, the solution was diluted with distilled water so that the final concentration of Tween 80 in the vehicle was 1% and the final concentration of RA ext was 0.1, 0.2, 0.3, and 0.4 mg/mL. The content of CO was 2.7% and DE was 2.3%. CO and DE were dissolved in a very small amount of methanol to ensure that methanol has no effect. In isolated intestine experiments, the solution was diluted with distilled water, and the final concentration of CO and DE was 5.4, 8.1, and 10.8 mg/mL and 4.6, 6.9, and 9.2 mg/mL, respectively. Drugs and reagents Neostigmine methylsulfate injection (2 mL: 1 mg), 5-HT, Ach, and verapamil were from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). CaCl2, MgCl2, KCl, NaHCO3, NaH2PO4, NaCl, NaOH, glucose, and trichloroacetic acid were produced by Tianjin Fengchuan Chemical Reagent Science and Technology Co., Ltd. (Tianjin, China). Stock solutions of all the chemicals were made in distilled water and the dilutions were made fresh in normal saline (0.9% sodium chloride) on the day of experiment. The power of RA ext was resuspended in 0.5% carboxymethyl cellulose (0.5% CMC). Vehicles used had no effect in control experiments. Standards including CO and DE were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All the reference compounds have over 98% purity. Methanol and acetic acid were both HPLC grade and ultrapure water was used for all analyses. Methanol of analytical grade was purchased from Guangfu Technology Limited Company (Tianjin, China). Instrumentations The HPLC apparatus was composed of LC-20AT (Shimadzu, Tokyo, Japan), a diode array detector (DAD) (SPD-M20A, Shimadzu, Tokyo, Japan), and column oven (CTO-20A, Shimadzu, Tokyo, Japan). The analytical column was a Kromasil C18 (250 mm  4.6 mm, 5 mm). All analyses were performed on an Agilent 1100 liquid chromatography system (Agilent Technologies, Santa Clara, CA), equipped with a quaternary pump, an online degasser, and a column temperature controller, coupled with an DAD (Alltech Associates, Deerfield, IL) as the detector. The analytical column was a Kromasil C18 (250 mm  4.6 mm i.d., 5 mm particle size) and the column temperature was kept at 35 C. The mobile phase was methanol and water (70:30, V/V) acidified with 2% acetic acid, at a flow of 1 mL/min. Animals Adult male and female KM mice (18–22 g) and male Wistar rats, weighing (250–270 g) (License no. SCXK (Jin)-2007004, Beijing Hongbo Kang Pharmaceutical Technology Co., Ltd., Beijing, China) were provided by the Laboratory Animal Center of Health Science, Peking University, Beijing, China. They were maintained on a 12-h light–dark cycle in an environmentally controlled breeding room

DOI: 10.3109/13880209.2013.879601

(temperature 22–25 C, humidity 60 ± 5%) for 7 d. Housed in polycarbonate cages (10 animals in each cage) with white wood chips for bedding, the animals had free access to water, but before experiments, they were fasted for 24 h. This animal research was in compliance with the Institutional Animal Care and Use Committee of China, and followed institutional guidelines for animal welfare and experimental conduct.

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intraperitoneal injection of neostigmine, phenol red was administered after 15 min, 20 min after the above treatment, the mice were killed by cervical dislocation, each group was evaluated by comparing with the vehicle control group. Gastric emptying and small intestinal calculated as the method list on the former part. In vitro study

In vivo study

Tissue preparation

Gastric emptying and small intestinal motility in normal mice

After fasting for 12 h, rats were sacrificed by a blow to the back of the head and cervical dislocation, then the intestine from duodenum to colon was flushed out and put in 0  C Tyrode’ solution (composition in mM: NaCl 136.9, CaCl2 1.8, KCl 2.7, MgCl2 1.1, NaHCO3 11.9, NaH2PO4 0.4, and glucose 5.6). Then jejunum was prepared with approximately 2 cm length, and placed in the a 25 mL organ bath with Tyrode’s solution, bubbled with 95% O2 and 5% CO2, and maintained at 37  C. Tissues were mounted under an initial load of 1.5 g, and it took 20 min to equilibrate before the addition of any drug. Mechanical activities were recorded on a computerized data processing system through an isometric force transducer (Biology BL-410, Digital Network Interactive Laboratory Teaching System, Weifang, China).

We assessed gastric emptying and small intestinal transit in 24 h fasted mice according to the method of phenol red as performed by earlier workers (Amira et al., 2005). RA ext (0.1, 0.2, 0.3, and 0.4 g/kg), trimebutine maleate tablet (0.5 g/kg) as a positive control, or 0.5% CMC (10 mL/kg) as a vehicle control was administered intragastrically in a volume of 0.1 mL/10 g body weight. After 30 min, a liquid meal containing phenol red (50 mg) suspended in 1.5% CMC (100 mL) was also gavaged in a volume of 0.3 mL and 20 min later, animals were sacrificed and the amount of phenol red retained in the stomach was measured. In each experiment, another 10 mice as control were also killed immediately after administration of the liquid meal at zero time (the absorbance of phenol red was considered as standards). After cervical dislocation, the stomach and the small intestine were excised after ligature of the pylorus and the cardias. The stomach was cut into several pieces, and homogenized in 25 mL of 0.1 M NaOH. The suspension was allowed to settle for 60 min at room temperature, and 4 mL of the supernatant was added to 0.5 mL of trichloroacetic acid (33% w/v). After centrifugation at 3000 rpm for 10 min, the supernatant was added to 1 mL of 2 M NaOH, and the absorbance of the sample was determined at 560 nm with a type 1800 spectrophotometer. Gastric emptying was calculated according to the formula (gastric emptying % ¼ 1amount of phenol red recovered from test stomach/ average amount of phenol red recovered from standard stomachs  100). Immediately after the excision of the stomach, the whole small intestine was grossly freed from its mesenteric attachments, and its length (from the pyloric sphincter to the ileocecal junction) was measured. The intestine was opened at the level of the front of the test meal, which was revealed by a few drops of 0.1 M NaOH. The rate of intestinal transit was expressed as the ratio between the distance travelled by the test meal and the total length of intestine. Intestinal transit (%) was expressed by the following formula: intestinal transit % ¼ the distance traveled by the phenol red/the total length of the small intestine  100. This method was described by previous studies (Mule et al., 2010). Gastric emptying and small intestinal motility induced by neostigmine Sixty animals were randomly divided into six groups. Vehicle control group received 0.5% CMC suspension (10 mL/kg), positive control group received trimebutine maleate tablet (0.5 g/kg), RA ext treated groups were administered orally at the doses of 0.1, 0.2, 0.3, and 0.4 g/kg for 40 min prior to

Effect of RA ext on smooth muscle contractions of isolated jejunum When the preparation was equilibrated in Tyrode’s solution for 30 min, we confirmed that the vehicle (1% Tween 80), in a volume equivalent to that of MeOH extract, had no significant effects on spontaneous smooth muscle contractions of the isolated rat jejunum. Then RA ext (0.1, 0.2, 0.3, and 0.4 mg/mL) was added in organ bath to test the effect on the spontaneous contraction. To study the prokinetic mechanism preliminary, the effects of RA ext (0.1, 0.2, 0.3, and 0.4 mg/mL) on the longitudinal muscle contractions induced by pretreatment of jejunum strips with Ach (105 M), 5-HT (200 mM) and KCl (60 mM) were investigated. Effects of RA ext on CaCl2-induced contraction To determine the effect of extract on Ca2+ influx, the preparation was initially equilibrated in Tyrode’s solution, and then Tyrode’s solution was replaced with Ca2+-free high-K+ (60 mM) solution containing EDTA (0.1 mM) for 30 min in order to remove Ca2+ from the tissues. The preparation was equilibrated in Ca2+-free high-K+ (60 mM) solution for 30 min, then dose–effect curves of CaCl2 (3  105– 3  103 M) in the absence and in the presence of RA ext (0.1, 0.2, 0.3, and 0.4 mg/mL) and verapamil (0.025 mM) were established. The contraction induced by 3  103 M CaCl2 in the absence of RA ext and verapamil was regarded as 100% (Wang et al., 2006). Effect of CO and DE on smooth muscle contractions of isolated jejunum As the main constituents of Radix Aucklandiae, CO and DE play an important role in the pharmacological action. The content of CO and DE in Radix Aucklandiae extract

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was 2.7% and 2.3%, respectively. So we studied the effects on the spontaneous contraction, Ach (105 M), 5-HT (200 mM), and KC1 (60 mM)-induced contractions of rat-isolated jejunum (Wang et al., 2006). Effects of CO and DE on CaCl2-induced contraction We made an in-depth study of the effects of CO and DE on CaCl2-induced contraction. After stabilization in normal Tyrode’s solution, the tissue was put in Ca2+-free highK+(60 mM) solution for 30 min, then dose–effect curves of CaCl2 (3  105–3  103 M) in the absence and in the presence of CO (5.4, 8.1, and 10.8 mg/mL), DE (4.6, 6.9, and 9.2 mg/mL), CO–DE (5.4–4.6, 8.1–6.9, and 10.8–9.2 mg/mL), and verapamil (0.01 mM) were established. The contraction induced by 3  103 M CaCl2 in the absence of CO, DE, CO– DE, and verapamil was regarded as 100% (Wang et al., 2006). Data analysis All data are expressed as mean ± standard error of mean (SEM) or percentage and analyzed for statistical significance using a one-way analysis of variance (ANOVA) followed by Dunnett’s test (Dunnett & Goldsmith, 1993). Tests were performed using SPSS 17.0 system (SPSS Inc., Chicago, IL). p Value less than or equal to 0.05 was considered as statistically significant.

Results Gastric emptying and small intestinal motility in normal mice and neostigmine-induced mice At the different doses ranging from 0.1 to 0.4 g/kg, RA ext had little effect on the gastric emptying comparing with the vehicle control group in normal mice. However, in neostigmine-induced mice, which could obviously accelerate the phenol red in the small intestine propulsion, the strongest inhibitory effect of gastric emptying was 80.99 ± 8.73% (p50.05) (Tables 1 and 2). In our experiments, we could see evident inhibitory effect on small intestinal propulsion. As shown in Table 1, RA ext dose dependently inhibited the transit in normal mice, and the intestinal transit ratios were 51.49 ± 9.60, 46.10 ± 6.39, and 42.33 ± 10.87% (p50.01). In the vehicle group, the mean length of intestine traveled by the phenol red meal was 64.63 ± 10.54%. Compared with neostigmine-treated mice, RA ext reduced the mean length traveled by the phenol red meal like the trimebutine maleate, whose intestinal transit ratio was 55.54 ± 5.05% (Table 2).

Effects of RA ext on smooth muscle contractions of isolated jejunum RA ext caused a concentration-dependent relaxation of spontaneously contracting rat jejunum with an EC50 value of 0.947 mg/mL (0.1–0.4, 95% CI, n ¼ 5) (Figure 2). RA ext also exhibited inhibitory effect against Ach, 5-HT, and KC1-induced contractions in a dose-dependent manner, with respective EC50 values of 0.38, 0.234, and 0.617 mg/ mL. The corresponding maximum suppression ratios were 47.96 ± 2.44, 64.59 ± 6.50, and 34.89 ± 3.58% (Figure 3). Effects of RA ext on CaCl2-induced contraction The data showed that RA ext antagonized incompetitively the contraction of isolated jejunum strips of rats induced by cumulative concentration of CaCl2, and the effect increased with the increase of dosage. Just as verapamil (0.025 M), a well-known calcium antagonist, the extract (0.1, 0.2, 0.3, and 0.4 mg/mL) shifted the CaCl2 curves to the right and reduced the maximum contraction induced by 3  103 M CaCl2 to 70.21 ± 1.3, 49.93 ± 0.77, 32.00 ± 0.18, and 20.00 ± 0.60% (Figure 4). Effects of CO and DE on contractility of isolated jejunum strips of rats induced by Ach, 5-HT and KCl The data showed that DE had no obvious effect on intestinal smooth muscle contraction of rat jejunum; however, the compatibility of CO and DE showed an inhibiting effect. Take the normal tension as 100%, the tension changed after

Table 2. Effects of different doses of RA ext on gastrointestinal transit in neostigmine mice (n ¼ 8).

Treatment group Neostigmine Trimebutine maleate RA RA RA RA

Dosage (g/kg)

Gastric emptying (%)

Intestinal transit (%)

104 0.5 0.1 0.2 0.3 0.4

90.66 ± 3.85 79.25 ± 6.41* 87.90 ± 8.02 86.66 ± 13.13 82.55 ± 11.61* 80.99 ± 8.73*

86.21 ± 17.11 55.54 ± 5.05** 78.49 ± 12.41 70.78 ± 8.93* 74.74 ± 8.38* 75.71 ± 14.80

Compared with neostigmine mice: *p50.05 or **p50.01. One-way ANOVA followed by Dunnett’s t-test.

Table 1. Effects of different doses of RA ext on gastrointestinal transit in normal mice (n ¼ 8). Treatment group Vehicle RA RA RA RA

Dosage (g/kg)

Gastric emptying (%)

Intestinal transit (%)

CMC-Na 0.1 0.2 0.3 0.4

67.86 ± 11.04 73.25 ± 9.43 76.53 ± 11.25 74.19 ± 9.18 70.99 ± 9.67

64.63 ± 10.54 55.73 ± 6.68 51.49 ± 9.60** 46.10 ± 6.39** 42.33 ± 10.87**

Compared with normal mice: **p50.01. One-way ANOVA followed by Dunnett’s t-test.

Figure 2. Effects of different concentrations of RA ext on smooth muscle contraction of rat-isolated jejunum.

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Figure 3. Effects of different concentrations of RA ext on Ach (A), 5-HT (B), KCl (C)-induced contraction of rat-isolated jejunum. The contractions induced by Ach, 5-HT and KCl in the absence of the extract acted as control. Results are mean ± SEM, n ¼ 5. Significantly different from control ***p50.001.

adding drugs. They all can inhibit the contraction induced by Ach, 5-HT, and KCl, but the inhibition of Ach and 5-HT is higher than KCl (data not shown). Effects of CO and DE on CaCl2-induced contraction In this study, to confirm whether CO and DE had synergistic effect, their effects on CaCl2-induced contraction were studied. As the dosage increased, their antagonized effects on the contraction of isolated jejunum strips of rats induced by cumulative concentration of CaCl2 decreased, while their compatibility increased (Figure 5). Then, we compared their effects at the same concentration and found that the antagonized effect of DE was stronger than CO’s at 2, 3, and 4 mM (Figure 6).

Discussion

Figure 4. Dose–effect curves of CaCl2 on rabbit-isolated ileum in the absence (^) and in the presence of RA ext (m 0.1 mg/mL;  0.2 mg/ mL; — 0.3 mg/mL;  0.4 mg/mL) and verapamil (g 0.025 mM). Results are mean ± SEM, n ¼ 5.

Weichang’an Pill, a traditional Chinese medicine (TCM) formula, has been used to treat irritable bowel syndrome and functional diarrhea for several decades (Hu et al., 2009; Liu et al., 2013; Wang et al., 2012). As a main component in Weichang’an pill, RA accounts for 25% of the prescription. Obviously, RA plays an important role in it. There were some

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Figure 5. Dose–effect curves of CaCl2 on rabbit-isolated ileum in the absence (^) and in the presence of RA ext, costunolide, and dehydrocostuslactone. (A) RA ext (m 0.2 mg/mL), costunolide ( 0.54 mg/mL), dehydrocostuslactone (— 0.46 mg/mL), costunolide–dehydrocostuslactone ( 0.54–0.46 mg/mL), and verapamil (g 0.01 mM). (B) RA ext (m 0.3 mg/mL), costunolide ( 0.81 mg/mL), dehydrocostuslactone (— 0.69 mg/ mL), costunolide–dehydrocostuslactone ( 0.81–0.69 mg/mL), and verapamil (g 0.01 mM). (C) RA ext (m 0.4 mg/mL), costunolide ( 1.08 mg/mL), dehydrocostuslactone (— 0.92 mg/mL), costunolide–dehydrocostuslactone ( 1.08–0.92 mg/mL), and verapamil (g 0.01 mM). Results are mean ± SEM, n ¼ 5.

pharmacological research on the spasmolytic activity of RA (Godfraind et al., 1986; Gupta & Ghatak, 1967). In the present study, RA ext had no significant effect on gastric emptying in normal mice, but it had an inhibitory effect on gastric emptying in mice under neostigmine treatment. The organization of gastric emptying is complex and involves the coordination of motor activity in the proximal stomach, the antrum, the pylorus, and duodenum, as well as passive forces generated by intragastric volume and gravity (Horowit et al., 1994; Moran et al., 1999). The tonus of the sphincter is a determinant factor of gastric emptying rate (Ishiguchi et al., 2000).

As food is swallowed, the gastric fundus relaxes to accommodate the incoming nutrients. This receptive is termed as relaxation, which is coordinated by vagal efferent activity via noradrenergic and non-cholinergic mechanisms. As swallowing during the meal continues, the fundic filling and relaxation continue with little increase in intraluminal pressure. Gastric distention, activation of mechanoreceptor, and stretch receptors stimulate vagal afferent nerve activity, which in turn modifies vagal efferent traffic. The emptying of solid foods is accomplished by complex interplays among intragastric pressure, gastric peristalses, pyloroduodenal resistance, and neuroendocrine responses elicited by

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Figure 6. Dose–effect curves of CaCl2 on rabbit-isolated ileum in the absence (^) and in the presence of costunolide and dehydrocostuslactone. (A) Costunolide (m 2 mM), dehydrocostuslactone ( 2 mM), and verapamil (g 0.01 mM). (B) Costunolide (m 3 mM), dehydrocostuslactone ( 3 mM), and verapamil (g 0.01 mM). (C) Costunolide (m 4 mM), dehydrocostuslactone ( 4 mM), and verapamil (g 0.01 mM). Results are mean ± SEM, n ¼ 5.

the specific components of the particular meal (Zhang et al., 2005). Then, neostigmine treatment induced a significant increase of gastric emptying, confirming the importance of the cholinergic pathway in controlling the flow from the stomach to the duodenum. So, the above results indicated that RA ext may alleviate stomach function by influencing the release of endogenous Ach from autonomic neurons. Meanwhile, RA ext could inhibit the intestinal transit obviously. Propulsive motility is termed peristalsis and subserved by a complex pattern of neural reflexes that aim to relax intestinal muscle downstream (descending inhibitory reflex) and contract the muscle upstream (ascending excitatory reflex) of the intestinal bolus. Intestinal transit is controlled by both neural and myogenic mechanisms (Huizinga et al., 1998). Several mediators and

neurotransmitters govern these motor patterns. Ach is the main excitatory neurotransmitter in the enteric nervous system, whereas NO is the major transmitter of the inhibitory motor neurons (Waterman & Costa, 1994). Acetylcholinesterase inhibitors such as neostigmine cause an increase in the cholinergic activity in the gut wall and thereby increase contractile activity. Parasympathetic and intrinsic neurons of the enteric nervous system have been shown to release Ach, which results in an increased contractile activity of the gut wall (Burks, 1995). Ach released into the synaptic gap is cleaved by acetylcholinesterase, which is reversibly inhibited by neostigmine (Snape et al., 1997; Wilkins et al., 1997). In the present investigation, since the intestinal transit was inhibited by RA ext in normal mice and neostigmine-induced mice, we supposed this could be related to relaxation of the

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contractile activity of the smooth muscle layers, and investigated the effect of RA ext on the contractile activity of the smooth muscle layers. Major advances have been made in our understanding of the nervous system in the gastrointestinal tract, namely the enteric nervous system, which is found in the walls of the entire gastrointestinal tract from the oesophagus to the anus and associated glands (salivary glands and the pancreas) and the gallbladder (Hansen, 2003c). The enteric nervous system regulates gastrointestinal functions including motility (Hansen, 2003a), secretions (Cooke, 2000; Hansen, 1995), blood flow (Hansen et al., 1998), and the immune system (Frieling et al., 2000; Shanahan, 1998). In gastrointestinal motility, the smooth muscle cells form an electrical syncytium that is innervated by hundreds of excitatory and inhibitory motor neurons. Among them, excitatory stimuli are exerted by tachykinins (for instance, substance P), Ach, and 5-HT (Hansen, 2003a; Kunze & Furness, 1999; Olsson & Holmgren, 2001). Indeed, the enteric nervous system is a potential target for pharmacological treatment of gut motor disorders. Motility-modifying drugs include antispasmodics (e.g., gut-selective muscarinic type 3 receptor antagonists, calcium-channel blockers, and opiate receptor agonists), prokinetics (e.g., serotonergic drugs), cholinergics (e.g., neostigmine), somatostatin analogues (e.g., octreotide), and antibiotics for bacterial overgrowth (Hansen, 2003b). In the previous study, the total lactone, CO, and DE of Saussurea lappa possessed relaxant effect on isolated rabbit duodenum, and they could reduce bronchospasm induced by the histamine and Ach aerosol in guinea pig (Gupta & Ghatak, 1967). As our experiment showed, RA ext exhibited a concentration-dependent spasmolysis effect on the isolated rat jejunum, which may explain the deceleration of intestinal propulsion found with the in vivo study. According to a previous study (Brunton, 1996), the property of reducing intestinal contractions is demonstrated by most antidiarrheal agents, and this helps in preventing excessive loss of fluid and ingesta. Furthermore, the experimental results (Figure 3) indicated that RA ext significantly abolished the contractility of isolated tissue preparations of rat jejunum treated with Ach and 5-HT. Therefore, we could infer that the regulatory of effects of the extract on GI motility is mediated via both muscarinic and 5-HT receptor. Besides neural factors, the myogenic factor is also included in the study on the smooth muscle contractions. K+ at high concentrations (430 mM) is known to cause smooth muscle contractions through the opening of voltage-dependent Ca2+ channels (VDCs), thus allowing an influx of extracellular Ca2+ causing a contractile effect and substances causing inhibition of high K+-induced contraction are considered to be blockers of calcium influx (Gilani et al., 2007; Godfraind et al., 1986). In order to assess whether the spasmolytic activity of RA ext works based on a calcium channel blockade, a high concentration of K+ (60 mM) was used to obtain a sustained contraction, allowing concentrationdependent inhibitory response data to be obtained. To confirm the calcium channel blockade, the contraction of smooth muscle preparations is dependent on an increase in the cytoplasmic-free Ca2+, which activates the contractile

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elements (Karaki & Weiss, 1988). The increase in intracellular Ca2+ is due to either influx via VDCs or release from intracellular stores in the sarcoplasmic reticulum. The results showed that the extract shifted the CaCl2 dose–effect curves to the right in a non-competitive manner, similar to that caused by verapamil, a standard calcium channel blocker (CCB) (Yang et al., 2007). So it was suggested that the extract was acting as an antagonist of either neurotransmitter to block their effect by preventing the release of Ca2+ from the cisternae, and hence it enters into the cell to activate smooth muscle contraction (Horowitz et al., 1996). Similar to the previous study, the aqueous-methanol crude extract of the Saussurea lappa root with the spasmolytic effect was marked in the spontaneously contracting rabbit jejunum and in the atropinized preparations, which were mediated through calcium channel blocking activity (Gilani et al., 2007). Phytochemical studies showed that R. Aucklandiae contained coumarins, flavonoids (Jia et al., 1983), and sesquiterpene lactones. CO and DE with a guaiane skeleton belonging to sesquiterpene lactones were the major active constituents in RA (Li et al., 2005). Interestingly, in the following study, we found that the compatibility of CO and DE had the synergistic effect as a calcium antagonist in Ca2+-free solution. Consequently, as the main constituents, their synergistically interactions may be one of the reasons for contributing to the spasmolysis of RA ext. Besides, the results indicated that the effect of DE as calcium antagonist was stronger than CO at the same concentration, which should be investigated by further study.

Conclusions In conclusion, the present results suggested that the gastrointestinal dynamic mechanism of RA ext might be the main inhibitory activity. Moreover, in vitro, RA ext possesses smooth muscle relaxing effect on high K+- and 5-HT, Ach-induced contractions of rat-isolated jejunum, mediated possibly through the combination of Ca2+ antagonist and anticholinergic mechanisms, which provide scientific basis for the clinical use of R. Aucklandiae in gastrointestinal disorders. In summary, the identification of the chemical components in RA ext and its metabolites in rats should be performed in further study.

Declaration of interest We have no conflict of interest in this research. The work was supported by the Natural Science Foundation of Tianjin, China (No. 13JCQNJC13000).

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