Liangshun Cao, Tianyu Liu, Chunhui Cai, Yan Li and Chongming Wu(.College of Life Science, Beijing Institute of Technology, Beijing 0008, China; .Fujian Chunhui Bioengineering Co., Ltd., Ningde 3500, Fujian, China; 3.Pharmacology and Toxicology Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 009 China)

With the continuous development of social economy and tremendous changes in lifestyles, constipation has become a common disease in modern society. Reduced amount of stool, extended defecation interval, defecation unawareness and defecation difficulties are main clinical symptoms of constipation[1-4]. There are three main types of clinical constipation: slow transit constipation, outlet obstruction constipation and mixed constipation. Among the three types of constipation, the slow transit constipation is most prevalent. Camellia oil is a unique woody oil found in China. It has proven benefits to preventing cardiovascular sclerosis, lowering blood pressure, decreasing blood lipids, etc[5-11]. In South China, camellia oil is used as a folk medicine to relieve constipation. So far, there is still lack of report verifying the constipation ameliorating effect of camellia oil via modern pharmacological design. In this work, two costive mice models induced by water deprivation and sucralfate were used to evaluate the alleviating effect of camellia oil on constipation. The potential mechanism was also investigated.

1 Materials and Methods

1.1 Animals and reagents

Camellia oil was provided by Fujian Chunhui Bioengineering Co., Ltd. (Ningde, Fujian, China). Hemp seed oil was purchased from Guangxi Bamadiyao Healthy Food Co., Ltd. (Bama, Guangxi, China). Sucralfate tablets were provided by Shandong Renhe Tang Pharmaceutical Co., Ltd. (Ju’nan, Shandong, China). Biochemical kits for serum nitric oxide (NO) and nitric oxide synthase (NOS) were gotten from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The kits for serum vasoactive intestinal peptide (VIP) and substance P were bought from Shanghai Yubo Biotechnology Co., Ltd. (Shanghai, China). Other chemical and biological reagents were from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

9-week old male BALB/c mice, weighing 25-30 g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were housed in the SPF animal workshop at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College. The room temperature was set to 22-24 ℃ with relative humidity of 50%-70% and a 12 h/12 h light/dark cycle. Food and water were availableadlibitum. All animal experiments were approved by the Medical Ethics Committee of Peking Union Medical College and were in accordance with the National Institutes of Health regulations for the care and use of animals in research. All efforts were made to minimize suffering.

1.2 Water deprivation-induced costive mice model

Sixty mice were randomly divided into six groups with ten animals in each group. The normal control (NC) group and model control (Model) group were given equal volume of distilled water. The positive control (PC) group was administrated hemp seed oil(4 mL/kg) daily. The low-, medium- and high-dose groups (camellia-L, camellia-M and camellia-H) were given camellia oil (2.0 mL/kg, 4.0 mL/kg and 8.0 mL/kg, respectively). Animals were treated using the respective agent for 5 days. Then, all mice, except the NC group, were banned from water with food availableadlibitumfor 3 days. During that time, each animal was given 0.3 mL of distilled water along with respective treating agent at 9 a.m. to meet the minimal living requirement. On the fourth day, all animals were fasted for food and water overnight, and then were given 0.2 mL of charcoal powder solution. The time interval for the first black feces defecation, total feces grain number and the total feces mass within 6 hours were recorded for each animal. On the fifth day, intestinal propulsion test was performed as described below. At the end of the experiment, animals were euthanized, whole blood was taken and serum was separated by centrifuge at 3 500 r/min for the measurement of serums NO, NOS, VIP and substance P by specific kits.

1.3 Sucralfate-induced costive mice model

Sixty mice were grouped and treated as described above. After treatment for 5 days, all mice except the NC group were given sucralfate (200 mg/kg,p.o.) along with respective reagent for 3 days. On the fourth day, all animals were fasted for food and water overnight and then were given 0.2 mL of charcoal powder solution. The time interval for the first black feces defecation, total feces grain number and the total feces mass within 6 hours were recorded for each animal. On the fifth day, intestinal propulsion test was performed as described below. At the end of the experiment, animals were euthanized, whole blood was taken and serum was separated by centrifuge at 3 500 r/min for the measurement of serums NO, NOS, VIP and substance P by specific kits.

1.4 Intestinal propulsion test

The animals were fasted for 8 hours before intestinal propulsion test then they were given the respective agent. Twenty minutes later, each mouse was administrated 2% phenol red (0.2 mL/animal). After another 30 min, orbital blood was taken and animals were euthanized by cervical dislocation. The digestive tract from cardia to rectum end was picked out and laid on a glass plate. The whole length of the small intestine (from the pylorus to the cecum) (LSI) and the distance of phenol red front to cardia (the length of phenol red propulsion) (LPC) were recorded. The intestine propulsion rate (IPR) was calculated as follows: IPR=LPC/LSI.

1.5 Statistical analyses

The results were presented as Mean±SEM. All data were statistically analyzed by SPSS 17.0 for Windows (SPSS, Chicago, USA).P<0.05 was considered statistically significant.

2 Results

2.1 Ameliorating effect of camellia oil on costive mice

The costive mice induced by the water deprivation or sucralfate showed obviously extended defecation time (Figs.1a and 2a), fewer feces grains(Figs.1b and 2b) less total stool mass (Figs.1c and 2c) and reduced fecal water content (Figs.1d and 2d). On both constipation models, administration of camellia oil dose-dependently reduced defecation time, increased defecation amount and enhanced fecal moisture (Fig.1 and Fig.2). The efficacy of camellia oil (8 mL/kg) was comparable to that of hemp seed oil (4 mL/kg), exhibiting an adequate laxative effect. Generally, camellia oil was more effective for constipation induced by water deprivation over that by sucralfate.

2.2 Effects of camellia oil on intestine propulsion

The transit defection resulting from the weakened gastrointestinal motility is the major mechanism for constipation incidence. The intestine propulsion test showed that the intestine motility was dramatically reduced in costive mice induced by either water deprivation or sucralfate (Fig.3 and Fig.4). Pretreatment with camellia oil (8 mL/kg) significantly enhanced the intestinal propulsion rate in both constipation models. Lower doses of camellia oil also exhibited alleviating but nonsignificant effects (Fig.3 and Fig.4).

Fig.2 Effects of camellia oil on costive mice induced by sucralfate(###P<0.001, NC vs Model, *P<0.05, **P<0.01, ***P<0.001, test group vs Model group)

Fig.3 Effects of camellia oil on intestine propulsion in costive mice induced by water deprivation ( ###P<0.001, NC vs Model, *P<0.05, **P<0.01, ***P<0.001, test group vs Model group)

Fig.4 Effects of camellia oil on intestine propulsion in costive mice induced by sucralfate(###P<0.001, NC vs Model, *P<0.05, **P<0.01, ***P<0.001, test group vs Model group)

2.3 Effects of camellia oil on key factors forintestinal motility

NO, NOS, VIP and substance P are key factors indicating intestinal motility. As shown in Fig.5 and Fig.6, the serum levels of NO, NOS and VIP were significantly increased in costive mice while substance P was decreased in both constipation models induced by water deprivation or sucralfate. Treatment with camellia oil (8 mL/kg) significantly shifted the serum levels of these factors to normal levels. Serum NO, NOS and VIP were largely decreased while serum substance P was significantly increased. The regulating efficacy of camellia oil (8 mL/kg) was comparable to that of hemp seed oil (4 mL/kg).

Fig.5 Effect of camellia oil on key factors of intestine propulsion in costive mice induced by water deprivation(###P<0.001, NC vs Model,*P<0.05,**P<0.01,***P<0.001, test group vs Model group)

Fig.6 Effect of camellia oil on key factors of intestine propulsion in costive mice induced by sucralfate (###P<0.001, NC vs Model,*P<0.05,**P<0.01,***P<0.001, test group vs Model group)

3 Discussion

Constipation is a global disease characterized by infrequent stool, hard texture, defecation difficulties and defecation awareness disappear. Slow transit constipation is the most common type of constipation in clinics, with an incidence of about 2%-27%. Epidemiologic investigation showed that incidence of slow transit constipation in Beijing is 6.07% and it rises up to 11.5% in the elders over 60-years of age[12-14]. Constipation, especially slow transit type constipation, can seriously affect the life quality of patients and has become an important social problem.

In this study, two constipation models were used. The costive model established by water deprivation mimics constipation caused by chronic insufficient water intake, while sucralfate treatment represented drug-induced constipation. Sucralfate is an approved drug used to treat chronic gastritis. It attaches to the gut mucosal surface, reduces the liquid in the gastrointestinal tract and inhibits intestine motility, which ultimately leads to constipation. Both models are similar to the symptoms of slow transit constipation. The results showed that administration of camellia oil (4 mL/kg and 8 mL/kg) can significantly shorten the defecation time, increase feces amount and enhance fecal moisture content, exhibiting good laxative effects with efficacy equivalent to hemp seed oil, a commonly used agent to embellish aperient bowel[11]. Therefore, camellia oil is a promising agent for the treatment of constipation.

Defecation is a physiological process that requires multiple systems and organs to function coordinately.Any malfunction in this system will inhibit defecation. The enteric nervous system is the main regulating system for defecation, which secrets excitatory neurotransmitters and inhibitory neurotransmitters to regulate the function of the digestive tract. Substance P is an important excitatory neurotransmitter. It inhibits gastrointestinal mucosa secretion, reduces water and electrolyte transport in gastrointestinal mucosal, and stimulates intestinal motility. Vasoactive intestinal peptide (VIP) is a major inhibitory neurotransmitter in the enteric nervous system, which is involved in the regulation of intestinal smooth muscle and vascular smooth muscle relaxation. Nitric oxide (NO) and its producer, nitric oxide synthase (NOS), are also major inhibitory factors for intestinal motility. Our results showed that camellia oil significantly enhanced the intestinal propulsion in costive mice induced by either water deprivation or sucralfate. It significantly reduced the serum levels of NO, NOS and VIP and increased serum substance P. These results suggested that the laxative effect of camellia oil might be achieved by regulating the level of key gastrointestinal motility factors and thereby enhancing intestinal propulsion.

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