Constipation is a common gastrointestinal disorder, which is a more complex pathogenesis, based on mainly gastrointestinal disturbance. The clinical manifestations of constipation are defecation difficulties, prolonged defecation time and so on. The classification of organic disease constipation can be divided into organic and functional . Constipation has a tremendous impact on the life quality of patients, and even seriously threatens the lives of patients. The incidence of constipation in China is 6% and the overall prevalence is higher in females than in males [2-4].
Polysaccharides, as informative molecules, have been found to play an important role in growth and development, cell recognition, antiviral, anticoagulant, immune regulation and the maintenance of immune system balance [5-8]. Previous studies show that polysaccharides have some therapeutic effect on constipation such as Long C.X. et al.  found that Dendrobium candidum polysaccharide could enhance the body immunity, regulate the balance of intestinal microbiota and intestinal enzyme activity, optimize the intestinal environment and improve the symptoms of spleen constipation. Luo D.D. et al.  studies show that the polysaccharide of D. officinalis has the laxative activity to constipated mice. Li H. et al.  used Semen plantaginis crude polysaccharidescapsules to treat 93 cases of constipation patients, the results showed that S. plantaginis crude polysaccharides capsules could improve constipation. S. plantaginis polysaccharides and garlic polysaccharides extracts had laxative effect [12,13].
Malus halliana Koehne (Figure 1), belonging to the Rosaceae family, is distributed in Shanxi, Jiangsu, Anhui and Zhejiang Province in China . M. halliana, taste slightly bitter, astringent, ping, belonging to the liver meridian. M. halliana has been used as a traditional Chinese medicine to treat metrorrhagia . Pharmacological studies showed that M. halliana had the ability of protect liver, antioxidant, inhibition of α-glycosidase enzymes in vitro and antithrombotic effect in vivo [16-18]. Chemical composition research of M. halliana focused on flavonoids [19,20]. No studies on biological activity of polysaccharides have been reported. As a part of our continuous research for M. halliana flowers. In this study, the M. halliana flower’s polysaccharides were extracted and investigated on functional constipation.
2 Materials and Methods
Male Sprague-Dawley/specific pathogen free (SD/SPF) rats weighting 200-220 g were purchased from Henan Animal Experiment Center (with License key of SCXK 20150004) (Zhengzhou, Henan, China). All animal studies are conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
2.2 Chemicals and Plant Materials
Loperamide hydrochioride (batch number: 170421860) was obtained from Xian Janssen Pharmaceutical Ltd. (Xian, Shanxi, China). Maren pill (batch number: 20170203) was obtained from Wuhan Tai Fu Pharmaceutical Co., Ltd. (Wuhan, Hubei,China). Rat Motilin (MTL), Rat Gastrin (Gas), Rat Substance P (SP), Rat Somatostatin (SS), (Batch numbers: 201709) were obtained from Senbeijia Nanjing Biotechnology Co., Ltd. (Nanjing, Jiangsu,China).
M. halliana flowers were collected in the Jinming campus of Henan University (Kaifeng, China) in March, 2017 (The voucher Number: 2017032601) and identified by Professor Changqin Li (Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province). A voucher specimen was deposited in Joint International Research Laboratory of Food & Medicine Resource Fuction, Henan Province.
2.3 Extraction and isolation of polysaccharides
M. halliana flowers (960 g) were extracted by petroleum ether under 45˚C three times for 2 h each time and the residue was extracted three times with 70% ethanol for 24 h each time. The solvents were filtered for 3 h each time, and the residue was extracted three times with distilled water of 1:24 (w/v) at 90˚C. Then, the filtrate was filtered and concentrated under reduced pressure at 50˚C, the precipitated with 95% ethanol (the final concentration was 80%) for 24 h. The precipitate was drip washed with 100% ethanol, acetone, petroleum ether each leaching 3 times, then re-dissolved in distilled water and concentrated under reduced pressure at 50˚C. Finally, the polysaccharides solution was dehydrated by freeze-dried to obtain the crude polysaccharides.
2.4 Acute toxicity test
The results of the maximum dose test of M. halliana polysaccharides showed no behavioral abnormalities, apathy, convulsions, no change in skin and respiration, after the rats were observed for 3 days. The rat was dissected and its heart, liver and spleen were excised and no change was observed with the naked eye. So, M. halliana polysaccharides did not cause visible toxicity.
2.5 Experimental Design
2.5.1 Establishment of Constipation Model with loperamide hydrochioride
Forty eight SD rats were kept for a week (Room temperature 25±2˚C, light for 12 h/d, relative humidity 40 ~ 45%). According to body weight, all rats were randomly divided into six groups each consisting of 8 animals: normal group, model group, positive control group, M. halliana polysaccharides high dose group, medium dose group and low dose group, respectively. Three days before the model establishment, each group of rats were collected 24 h-feces, weighed wet weight, dried 8 h at 50˚C. Wet and dry granulation rat weight was recorded before model establishment.
The normal group was orally administered with normal saline daily, the other groups were given intragastric administration with loperamide hydrochloride (3 mg / kg·d·2 mL ) according to weight.
When the rats showed poor mental status, eating less, weight loss, stool grain size, dry and hard performance, the success of modeling for 14 d.
During the establishment of the rat constipation model, the rats were collected regularly for 24 h. The wet weight of the pellets was weighed, placed in an electric oven, dried at 50˚C for 8 h, and the pellets were weighed. The rats were weighed one day apart and the data was recorded. Rats shrimp shape and rat activity in each group was observed.
2.5.2 Treatment with M. halliana polysaccharides
The rats in the normal group and the model group received 1 mL/100 g normal saline daily. Rats in the positive control group received Maren pill 0.6 g / kg, 1 mL / 100 g for gavage. Rats in the M. halliana polysaccharides high dose group, medium dose group and low dose group received 1200, 1000 and 800 (mg/kg), 1 mL/100 g weight of M. halliana polysaccharides, respectively.
During the treatment of rats, their 6 h shit were collected regularly every day, weighed and the data recorded. After 7 days, rats in each group were fasted for 12 h, then anesthesia with 10% chloral hydrate, and abdominal aorta blood was collected, 30 min at room temperature and centrifuged at 3000 r / min for 10 min to obtain rats serum samples were collected and the content of MTL, GAS, SS and SP were measured. The colon of the lower part of cecum was excised 2 cm, washed with tap water and saline, fixed with 4% paraformaldehyde for 24 h, 10 μm section, HE staining; 2 cm away from the cecum colon 2 cm, saline clean, filter paper dry, into the oven, 50˚C drying 24 h.
2.6 Statistical analysis
All experimental results were expressed as mean ± standard deviation (SD). Statistical analysis was performed with the SPSS19.0. Comparison between any two groups was evaluated using one-way analysis of variance (ANOVA).
3.1 Effect on defecation in rats
Compared to the normal group, activities and diets of rats in each model group were decreased, furred coat color and easy hair removal, and the defecation of rats in model group decreased. In Table 1, compared to the normal group, the model group (p <0.001) 24 h shrimp weight significantly decreased, indicating that constipation model was established.
In Table 1, compared to the normal group, the weight of the model group (p <0.001) 6 h was significantly decreased than that of the treatment group and the normal group, which indicated that the constipation symptoms of the model group did not alleviate. Compared to the model group, the weight of the positive control group (p <0.001), the high, medium and low (p <0.001) doses of M. halliana polysaccharides significantly increased. Compared with the positive control group, the weight of M. halliana polysaccharide high, medium dose groups was similar and the effect was similar, the low dose group (0.01 < p <0.001) significantly increased and the effect was better than that of the positive control group.
3.2 Effect on rat colonic moisture content
Compared to the normal group, the model group (p <0.001) significantly decreased the water content of the colon, indicating that the model group did not reduce the symptoms of constipation. Compared with the model group, the water content of colon in the positive control group (0.001 < p <0.01) and the low of M. halliana polysaccharides (p <0.001) significantly increased and the colon water content significantly increased. Compared with the positive control group, the colon water content of the high, medium and low dose of M. halliana polysaccharide was similar to that of the positive control group.
3.3 The levels of motilin (MTL), gastrin (GAS), substance P(SP) and somatostatin (SS)
As shown in Table 3 and 4, compared with the normal group, the contents of GAS, MTL and SP in the model group were significantly decreased (p <0.001), and the SS content was significantly increased. Compared to the model group, the content of GAS and MTL in the positive control groups, high dose group, medium dose group and low dose group significantly increased (p <0.001). Compared to the model group, the contents of SP in the positive control groups, medium dose of M. halliana polysaccharides significantly increased, the levels in the low dose group significantly increased, and those in the high dose group were increased. Compared with the model group, the contents of SS in the positive control group, M. halliana polysaccharides high, medium and low dose group significantly decreased.
The results showed that the positive control group, M. halliana polysaccharides high, medium and low dose group had a therapeutic effect on constipation.
3.4 Histological observations
From the histological sections HE staining, the normal group (Figure 2a) cells in normal morphology, cells arranged closely, and the model group (Figure 2b) increased cell matrix, irregular shape. Compared with the model group, the positive control group (Figure 2c), M. halliana polysaccharides high dose group (Figure 2d), medium dose group (Figure 2e), low dose group (Figure 2f), the cell morphology was restored, the cell matrix was reduced.
Clinical manifestations of functional constipation mainly defecation effort, fecal dry knot, a sense of defecation and anorectal obstruction or obstruction can also be manifested as reduced the amount of defecation and frequency, which not only affects the appetite and intestinal absorption of patients, and can induce or aggravate colon cancer, cardiovascular and other diseases. This topic based on pre-experimental and reported in the literature [21,22], the choice of 3 mg / kg loperamide hydrochloride suspension twice daily gastric perfusion model, rats with poor mental status, reduce eating, weight loss, stool grain size small, dry hard and other clinical manifestations of constipation. There is a variety of gastrointestinal hormones in the human gastrointestinal tract to regulate gastrointestinal motility, which has important physiological significance, such as motilin (MTL), gastrin (GAS) and somatostatin (SS) [23-25]. Intestinal neurotransmitters are also involved in the regulation of gastrointestinal motility, such as substance P (SP) . Researches show that motilin (MTL) is an important neurotransmitter of gastrointestinal excitatory motor neurons, can speed up the gastrointestinal motility, improve gastrointestinal contractility and tension. Gastrin (GAS) mainly secreted by gastric antrum, duodenum and small intestinal mucosa open G cells, and stimulates gastric acid secretion and promotes gastrointestinal motility. Somatostatin (SS) inhibits smooth muscle contraction and the secretion of gastrin and pepsin. Substance P (SP) can inhibit gastrointestinal mucosal secretion, stimulate intestinal motility and contraction of smooth muscle movement. In patients with constipation, the levels of motilin (MTL), gastrin (GAS) and substance P (SP) were significantly decreased, and somatostatin SS content was significantly increased. The effect of M. halliana polysaccharides on functional constipation was evaluated by using six indexes of defecation, water content of colon, MTL, GAS, SS and SP. The results showed that the three doses of M. halliana polysaccharides could improve functional constipation in rats, low-dose group was the best than the other two dose groups.
Numerous studies have been reported that many kinds of polysaccharides, such as algae and plants, have the effect of improving constipation, such as Enteromorpha polysaccharides, Inula japonica polysaccharides, Tremella polysaccharides and Flammulina velutipes polysaccharides [27-30]. The mechanism may be related to the structure of polysaccharides and gut microflora, but the specific Mechanism needs to be further studied.
This work was supported by the Henan Province University Science and Technology Innovation Team (16IRTSTHN019), Key Project in Science and Technology of Henan Province (182102110473).
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About the article
Published Online: 2018-10-22
Conflict of Interest: The authors declared no potential conflicts interest with respect to the research, authorship, and publication of this article.
Citation Information: Open Chemistry, Volume 16, Issue 1, Pages 956–962, ISSN (Online) 2391-5420, DOI: https://doi.org/10.1515/chem-2018-0107.
© 2018 Yingying Niu et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0