Yi-Sook Jung and Mi-Young Kim contributed equally to this work.
This work has been previously published as an abstract at the Joint International Neurogastroenterology and Motility Meeting, 2012.
Address for Correspondence Kwang J. Lee, Department of Gastroenterology, Ajou University School of Medicine, Ajou University Hospital, 164 Worldcup-ro, Yeongtong-gu, Suwon 443-721, Korea. Tel.: +82 31 219 5102; fax: +82 31 219 5999; e-mail: email@example.com
Background DA-9701 is a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber. This study aimed to evaluate the effect of DA-9701 on stress-induced delay in gastric emptying and changes in plasma adrenocorticotropic hormone and ghrelin levels in rats.
Methods Changes in gastric emptying in response to different durations of stress were evaluated. Gastric emptying was compared between the following groups: (i) nonstressed vehicle- or DA-9701-treated group, (ii) nonstressed vehicle- or mosapride-treated group, (iii) 2-h stressed vehicle- or DA-9701-treated group, and (iv) 2-h stressed vehicle- or mosapride-treated group. Water immersion restraint stress was used as the stressor. DA-9701 or mosapride at 3 mg kg−1 was administered to the rats after subjecting them to 2-h stress, and then gastric emptying was measured using the phenol red method.
Key Results Gastric emptying was significantly delayed in the 2-h stressed group compared with the nonstressed group. Mosapride administration resulted in significant recovery from the stress-induced delay in gastric emptying. Gastric emptying in the rats that underwent 2-h stress followed by DA-9701 administration was not significantly different from that in the nonstressed group. The plasma adrenocorticotropic hormone and active ghrelin levels in the 2-h stressed group were significantly higher than those in the nonstressed group. These increases were significantly inhibited by DA-9701.
Conclusions & Inferences The administration of DA-9701 improved delayed gastric emptying and inhibited the hormonal changes induced by stress, suggesting that DA-9701 can be used as a gastroprokinetic agent for the treatment of delayed gastric emptying, particularly that associated with stress.
Stress has been shown to induce or exacerbate dyspeptic symptoms.1,2 Stress may alter gastric sensory and motor functions. Acute stressors have been shown to delay gastric emptying in experimental animals and healthy humans.3,4 Furthermore, stress increases visceral sensitivity, leading to enhanced perception of gut stimuli.5 Increased severity of dyspeptic symptoms induced by acute stress in patients with postprandial distress syndrome was found to be associated with an enhanced sympathetic activity and increased serum levels of stress hormones, including adrenocorticotropic hormone (ACTH) and cortisol.6 The corticotrophin-releasing factor (CRF) appears to play a crucial role in mediating stress-induced delay in gastric emptying. In particular, central and peripheral CRF signaling pathways are involved in stress-induced changes in gastric motility.3,4 Sympathetic neural pathways also contribute to stress-induced delay in gastric emptying. Plasma ghrelin levels have been reported to increase in rat models of stress.7,8 However, given that ghrelin is known to enhance gastrointestinal motility, its role in the gastric motor response to acute stress remains unclear.
Prokinetic drugs are commonly prescribed to dyspeptic patients with suspected delayed gastric emptying or dysmotility. However, the gastroprokinetic agents cisapride and tegaserod are associated with serious adverse effects,9 and the efficacy data on itopride and mosapride are conflicting.10,11 Therefore, the development of safer and more effective gastroprokinetic agents is warranted. DA-9701 is a newly developed prokinetic agent formulated with the extracts of Pharbitis Semen and Corydalis Tuber. These plants have been used in traditional Oriental folk medicines. Pharbitis Semen, the seed of Pharbitis nil Choisy, is believed to have analgesic effects on the abdomen. Corydalis Tuber, the root of Corydalis yahusuo W.T. Wang (Papaveraceae), is known to have analgesic and antiulcer effects.12–14 DA-9701 was found to not only significantly accelerate gastric emptying in healthy rats but also normalize delayed gastric emptying induced by apomorphine and cisplatin.15 Hence, we hypothesized that DA-9701 would normalize stress-induced delay in gastric emptying.
Thus, in this study, we aimed to evaluate the effect of DA-9701 on delayed gastric emptying and changes in plasma ACTH and ghrelin levels induced by stress in rats.
Materials and Methods
Preparation of DA-9701
DA-9701, the standardized extract of Pharbitis Semen and Corydalis Tuber, was prepared as previously reported.15 Briefly, the two herbs were mixed in a specific dry weight ratio (water content <10%) and extracted with 50% aqueous ethanol (EtOH) at room temperature for 48 h. After filtration, the aqueous EtOH extract was evaporated under reduced pressure to yield a brown extract. The quality of DA-9701 obtained was evaluated using the standard method involving quantitative high-performance liquid chromatography, as previously reported.15
Adult male Sprague-Dawley rats weighing 300–350 g were housed in individual cages under controlled temperature (22–24 °C) and illumination (12-h light/dark cycle starting at 6:00 am) conditions for at least 7 days before the experiment. The rats had ad libitum access to food and water. All the experiments were started at 9:00 am. The experimental procedures were performed in accordance with the guidelines of the institutional animal care and use committee of Ajou University.
Figure 1 shows the schematic diagram of the experimental protocol. The animals were fasted for 24 h before the experiments. The rats were immobilized in a restrainer and subsequently immersed up to the xiphoid process in a water bath maintained at 18 °C ± 1 °C, as previously described.16 The rats in the nonstressed group were placed in their home cages without exposure to any stress. The animals were orally administered with DA-9701, mosapride (Sigma, St. Louis, MO, USA) as a positive control, or 3% (w/v) hydroxypropylmethyl cellulose (Sigma) as a vehicle.17 In this study, DA-9701 was used at a dosage of 3 mg kg−1 on the basis of a previous report that showed that this dosage significantly restored cisplatin-induced delayed gastric emptying.15 The mosapride dose used in the present experiments was also chosen based on a previous study.18
The study protocol consisted of two different parts: one related to DA-9701, and the other related to mosapride. Because each part was performed on a different day, we included a control group for each part of the experiment. Gastric emptying data of the 2-h stressed vehicle-treated groups were slightly different in the two parts of the experiment, but the difference was not statistically significant (P =0.216).
Measurement of gastric emptying
The effect of DA-9701 on gastric emptying was determined using the phenol red method, as described previously.19 A 1.5-mL suspension of continuously stirred 1.5% methylcellulose (Sigma) containing phenol red (50 mg per 100 mL; Sigma) was administered to conscious rats via oropharyngeal intubation at 45 min after drug administration. Gastric emptying of methylcellulose, a semisolid meal, was measured. The selected volume of 1.5 mL corresponds to a single physiological meal in adult rats. After 1 h, the rats were killed by decapitation. The stomach was removed, rinsed in 0.9% saline, and homogenized in 100 mL of 0.1 mol L−1 NaOH. The homogenate was allowed to settle for 60 min at room temperature, and 5 mL of the supernatant was added to 0.5 mL of trichloroacetic acid solution (20%, w/v) and then centrifuged at 1600 × g at 4 °C for 20 min. The supernatant was mixed with 4 mL of 0.5 mol L−1 NaOH, and the light absorbance of the sample was recorded at 560 nm. Phenol red recovered from the stomachs of the rats killed immediately after oropharyngeal delivery of the methylcellulose solution served as the 0% emptying standard. The percentage of gastric emptying for the 1-h period was calculated according to the following equation: % gastric emptying = [(A560 standard − A560 sample)/A560 standard] × 100.
Measurement of plasma ghrelin and ACTH levels
Blood samples were collected by cardiac puncture before the animals were killed. Plasma was separated by centrifugation and stored at −70 °C. Plasma ghrelin (acylated) and ACTH levels were measured using an enzyme immunoassay kit (Phoenix Pharmaceuticals, Burlingame, CA, USA) according to the manufacturer’s instructions.
All the data were expressed as mean ± SEM. Comparisons between the groups were analyzed using the Student’s t-test or one-way analysis of variance. P <0.05 was considered significant.
Effect of DA-9701 on stress-induced delay in gastric emptying
As shown in Fig. 2, gastric emptying was significantly delayed at the 1.5- and 2-h time points in the stressed groups compared with the nonstressed group (42.2% ± 3.9% and 23.4% ± 4.8%, respectively, vs 64.9% ± 6%; P <0.01). The delayed gastric emptying induced by stress for 2 h (22.4% ± 4.2%) was reversed by the administration of DA-9701 (3 mg kg−1, p.o.) to 69.8 ± 8.6% (P <0.01) (Fig. 3A). Mosapride, the positive control, also reversed the stress-induced delay in gastric emptying (29.4% ± 2.6%) to 64.2% ± 2.1% (P <0.01) (Fig. 3B). In the nonstressed control group, gastric emptying was not significantly changed by the administration of 3 mg kg−1 (p.o.) DA-9701 (64.0% ± 3.8%vs 66.5% ± 3.4%; P >0.05) but was significantly increased by the administration of 3 mg kg−1 (p.o.) mosapride (72.3% ± 1.8%vs 86.3% ± 5.8%; P <0.05).
Effect of DA-9701 on the plasma ACTH levels
The plasma ACTH levels of the 2-h stressed vehicle-treated group were significantly higher than those of the nonstressed vehicle-treated group (118.7 ± 9.8 pg mL−1vs 30.8 ± 2.1 pg mL−1; P <0.01). The plasma ACTH levels of the 2-h stressed DA-9701-treated group were significantly lower than those of the 2-h stressed vehicle-treated group (78.1 ± 12.1 pg mL−1vs 118.7 ± 9.8 pg mL−1; P <0.05) (Fig. 4).
Effect of DA-9701 on the plasma ghrelin levels
The plasma ghrelin levels of the 2-h stressed vehicle-treated group were significantly higher than those of the nonstressed vehicle- treated group (233.6 ± 17.0 pg mL−1vs 117.5 ± 4.9 pg mL−1; P <0.01) The plasma ghrelin levels of the 2-h stressed DA-9701-treated group were significantly lower than those of the 2-hour stressed vehicle-treated group (156.1 ± 20.4 pg mL−1vs 233.6 ± 17.0 pg mL−1; P < 0.05) (Fig. 5).
Various types of mental or social stress may cause dyspeptic symptoms,1,2 possibly via the consequent alterations in gastric motor and sensory functions. Gastric emptying has been observed to be delayed during the acute phase of stress. In this study, we observed that delayed gastric emptying was induced by 1.5-h and 2-h water immersion restraint stress. The stress-induced delayed gastric emptying was normalized by DA-9701 treatment. The stress-induced increases in plasma ACTH and acylated ghrelin levels were significantly reversed by the administration of DA-9701. These findings suggest that DA-9701 can be considered as a gastroprokinetic agent and influences stress-induced hormonal changes.
DA-9701 has been commercially produced under the name Motiliton (Dong-A Pharm. Co., Seoul, South Korea) in South Korea since early 2012. DA-9701 is a prokinetic agent formulated from Pharbitis Semen and Corydalis Tuber extracts. Corydalis Tuber extracts exhibit antispasmodic and analgesic effects.12–14 Corydalis Tubers are composed mainly of alkaloid compounds, including corydaline, berberine, protopine, and palmatine,20,21 which are reported to have analgesic, antispasmodic, and anti-acetylcholinesterase activities. Corydaline, a marker compound used for the quality control of DA-9701, was reported to not only significantly accelerate gastric emptying but also induce significant gastric relaxation.12 DA-9701 has been observed to have an affinity for the D2, 5-HT4, 5-HT1A, and 5-HT1B receptors. It has antagonistic effects on the D2 receptors and agonistic effects on the 5-HT4, 5-HT1A, and 5-HT1B receptors.15,22,23
Kim et al.23 reported that DA-9701 administration had beneficial effects on gastric accommodation in conscious dogs. In their study, DA-9701 treatment significantly increased basal gastric volume and gastric accommodation to a meal in dogs. Furthermore, a previous animal study demonstrated the gastroprokinetic effects of DA-9701 in a model of delayed gastric emptying using apomorphine.15 In the study, gastric emptying was markedly delayed by the subcutaneous administration of apomorphine, which was restored by DA-9701 treatment. Because a maximal effect was observed in the rats treated with 3 mg kg−1 DA-9701, we used the same dose for our experiments. We used a model of delayed gastric emptying using water immersion restraint stress. Gastric emptying was markedly delayed by 2-h water immersion restraint stress to approximately 36% of that of the nonstressed control rats (23.4% ± 4.8%vs 64.9% ± 6%). The oral administration of DA-9701 at a dose of 3 mg kg−1 normalized stress-induced delayed gastric emptying (69.8% ± 8.6%vs 64.9% ± 6%). The results of our earlier optimization experiments indicated that the effect of DA-9701 on gastric emptying was maximal after 45 min of oral administration (results not shown); therefore, in this study, we evaluated the experimental parameters at 45 min after DA-9701 administration. Delayed gastric emptying induced by stress may be more likely similar to that observed in patients with functional dyspepsia than delayed gastric emptying induced by apomorphine or cisplatin. The DA-9701 treatment had no significant effect on gastric emptying in the nonstressed control rats, which is in contrast with an earlier report.15 This discrepancy in results is difficult to explain but may at least be partly attributed to the difference in the methodologies used, including the age and weight of the rats used, the stress duration and methods, and the timing and technique of gastric emptying measurement. In this study, the gastroprokinetic effect of DA-9701 was comparable with that of mosapride. Our results indicate that DA-9701 is capable of normalizing stress-induced delay in gastric emptying, suggesting that DA-9701 can be used as a gastroprokinetic agent for the treatment of delayed gastric emptying.
Corticotrophin-releasing factor and sympathetic neural pathways were found to be involved in stress-induced delay in gastric emptying.3,24 Corticotrophin-releasing factor-induced inhibition of gastric emptying is mediated via a peripheral sympathetic pathway.3 In this study, acute stress resulted in increased plasma ACTH levels, which were inhibited by DA-9701 administration. The mechanism underlying the inhibitory effects of DA-9701 on increased plasma ACTH levels remains to be elucidated. DA-9701 administration may normalize gastric emptying from stress-induced delay through inhibition of the CRF and sympathetic neural pathways.
Ghrelin, a gut peptide secreted from the stomach, not only stimulates food intake but also enhances gastric motor and secretory functions.25 Acute psychological stress has been reported to increase plasma ghrelin levels in rats.26 However, the pathway responsible for the alteration of plasma ghrelin levels under stress conditions still remains to be elucidated. Ghrelin molecules in the blood are present in two major forms: the acylated (active ghrelin or ghrelin) and des-acylated (desacyl ghrelin) forms. Acylation is considered to be important for most biological activities of ghrelin.8,27,28 Ghrelin exerts enhancing effects on the motility of the antrum and duodenum in animals. A previous study in a chronic stress rat model showed that plasma active ghrelin levels were significantly increased but plasma total and desacyl ghrelin levels were not.8 We measured plasma acylated ghrelin levels in this study and confirmed its significant increase in stressed rats. Moreover, this study showed that the administration of DA-9701 inhibited the increase in plasma acylated ghrelin levels induced by acute stress. Considering that the stress-induced increase in plasma ghrelin levels was associated with the response of serum cortisol levels to acute stress,29 the inhibitory effect of DA-9701 on the increase in plasma ghrelin levels might be associated with its inhibitory effect on the increase in plasma ACTH levels. Given that ghrelin enhances appetite, high plasma ghrelin levels may induce the urge to eat. However, a previous study has demonstrated that the urge to eat after stress is not acutely modulated by stress-related increases in ghrelin levels.29 Similarly, the elevation in plasma ghrelin levels observed in the acute stress rat model of this study did not seem to be involved in the acute change in gastric emptying. While continuous stress delayed gastric emptying until 24 h, gastric emptying was accelerated on days 3 and 5.7 The increase in plasma ghrelin levels was suggested to play a role in the acceleration of gastric emptying during the chronic phase of continuous stress.7 The results of this study showed that the stress-induced increase in plasma ghrelin levels was inhibited by the administration of DA-9701. The inhibitory effect of DA-9701 on increased ghrelin levels might reduce the influence of continuous stress on gastric emptying, body weight, or appetite, but this requires further investigation.
Besides ghrelin, other hormones or peptides could be involved in the stress-induced delay in gastric emptying. Glucagon-like peptide 1, leptin, and obestatin are capable of decelerating gastric emptying.30–32 Central somatostatinergic signaling pathways and oxytocin are suggested to be involved in gut motor responses to acute stress.33,34 Whether DA-9701 directly works on the gastric motility through a peripheral mechanism or controls the gastric motility through a central mechanism requires further study.
In conclusion, this study indicates that DA-9701 improved stress-induced delay in gastric emptying and inhibited stress-induced hormonal changes, suggesting that DA-9701 can be used as a gastroprokinetic agent for the treatment of delayed gastric emptying, particularly that associated with stress. Given that DA-9701 also improves gastric accommodation by increasing the postprandial gastric volume,23 DA-9701 may be an effective agent for postprandial distress syndrome, which is known to be associated with delayed gastric emptying or impaired gastric accommodation to a meal.
This study was supported by Dong-A Pharm. Co. The data of this study were permitted to be disclosed by Dong-A Pharm. Co.
The authors have no competing interests.
YSJ, MK, and KJL were involved in study concept and design, experiments, analysis and interpretation of data, and drafting of manuscript; KJL obtained funding, submitted the Institutional Review Board application for approval to conduct the study, supervised all research activities, and revised the manuscript; HSL and SLP maintained the research equipment, provided technical assistance, and helped with data acquisition and analysis.