Original papers
Chimonanthus salicifolius S. Y. Hu Extract improve constipation symptoms and regulate intestinal microbiota in mice
2023 年 29 巻 2 号 p. 101-112
詳細
2023 年 29 巻 2 号 p. 101-112
Chimonanthus salicifolius S. Y. Hu (C. salicifolius) is a unique medicinal plant of Magnolia in China, it could inhibit pathogens and prevent flu, which has been extensively applied for drinking tea. The purpose of this study was to analyze the main components of ethyl acetate extract of C. salicifolius (EAECS) and explore the effect of extract on loperamide-induced constipation in mice. The mice with constipation induced by loperamide were given different concentrations of C. salicifolius extract once a day for 14 days. The constipation-related parameters, stool particles, the time of the first black stool defecation and gastrointestinal (GI) transit rate were determined. In addition, the change of abundance and diversity in intestinal microbiota were analyzed. The results indicated that the main components in EAECS were rutin, nicotiflorin, quercetin and kaempferol. After administration, EAECS treatment relieved loperamide-induced constipation in mice, as evidenced by reduced defecation time and significantly increased GI transit rate, fecal particles and water content. At genus level, the extract also remarkably reduced Ruminococcus and increased the abundance of Lactobacillus and Bifidobacterium in intestinal tract of mice (all p < 0.05). These findings indicated that EAECS effectively improved loperamide-induced constipation in mice and could be considered as a candidate treatment for constipation.
Constipation, a common GI disorder, is characterized by difficult, incomplete or low frequency of defecation, prolonged intestinal emptying time, which causes physical discomfort to people of all genders such as abdominal distension and bloating. Clinical study analysis reported the worldwide pooled prevalence of constipation is showing an increasing trend (Mugie et al., 2011; Bharucha et al., 2017), the prevalence of childhood ranged from 0.7 % to 29.6 % and the morbidity of elderly adults almost reached 36 % (Koppen et al., 2018; Rao et al., 2016). The occurrence of constipation seriously affected the physical and psychological health, including anxiety and depression, even long-term constipation ggravated cardiovascular and cerebrovascular diseases (Sumida et al., 2019; Shapiro et al., 2021). Some of the previously reported digestive system dysfunction caused by constipation can lead to abnormal serum gastrointestinal hormones and hyperglycemia metabolic disorders, which increase the risk of diabetes and colorectal cancer (Conway et al., 2020; Guérin et al., 2016). Pharmaceuticals including osmotic agents, stimulant and purgative have been widely used to treat constipation (Lucak et al., 2021; Tappin et al., 2020). However, long-term intake could cause side effects such as overdependence, abdominal pain, and gaseous distention (Pannemans et al., 2020; Skvortsov et al., 2020). At present, it is recognized as the best treatment of constipation for increasing physical activity and improving intake of dietary fiber to change the intestinal environment (Zhao and Yu, 2016; Shulpekova et al., 2020).
Chimonanthus salicifolius S. Y. Hu, a kind of the most valuable medicinal plants in Eastern China, which belongs to the Calycanthaceae family and Chimonanthus Lindley genus (Liu and Fu, 2018; Liang et al., 2016). The genome of C. salicifolius is comprised of 820.1 Mb genomic sequence containing 36 651 annotated protein-coding gene (Lv et al., 2020). C. salicifolius leaves have been made into a drinkable herbal tea as a traditional medicine to antiviral by She nationality in China. The Compendium of Materia Medica recorded that C. salicifolius belonged to the homology of medicine and food as a precious plant could eliminate pathogens (Chen et al., 2017), relieve diarrhea symptoms and detoxify for hundreds of years in Eastern China (Chen et al., 2017). Ethanolic extracts of C. salicifolius has significant antimicrobial and antibiotic-mediating activity (Wang et al., 2016). Besides, combination between streptomycin and alcohol extracts from leaves of the plant have exhibited higher inhibition against Escherichia coli and Staphylococcus aureus (Wang et al., 2019). Although references indicated that the plant has excellent antimicrobial properties, less study with intestinal modulation and bacterial microbiota changes reported on the specialty of the species.
Intestinal microbiota, a complex and diverse microbial including bacteria, fungi and protozoa, which revealed essential roles in environment balance, human growth, immune regulation, metabolism and nutrient absorption as an important microecosystem (Rodriguez et al., 2021; Naseer et al., 2020). These microbiota and host symbiosis maintain the stability of the environment, when the structure and number of intestinal microbiota have changed, people existed gastrointestinal symptoms such as irritable bowel syndrome and constipation (Malard et al., 2020; Berumen, et al., 2021). In fact, intestinal microbiota exhibited the difference between patients with constipation and normal people by clinical study. Constipation have demonstrated that directly related to the imbalance of intestinal microbiota, the beneficial microbiota can reduce the incidence of constipation (Min et al., 2020; Oganezova and Medvedeva, 2020). Moreover, when the microbiota dysbiosis occurred, the number of anaerobic bacteria decreased significantly, pathogenic bacteria increased with changes of abundance such as Enterobacter, Enterococcus, Fusobacterium, etc. (Dan et al., 2020). The abundance and diversity of these species among intestinal bacteria are usually considered for a major factor affecting constipation (Quigley and Spiller, 2016; Dandan et al., 2016).
Accordingly, this study aims to (i) detect the main components of ethyl acetate extract of C.salicifolius; (ii) evaluate the therapeutic effect of extract against loperamideinduced constipation in mice by GI transit rate and stool parameters, (iii) explore the underlying mechanisms by analyzing changes in abundance of intestinal microbial.
Materials and animals The leaves of Chimonanthus salicifolius S. Y. Hu were obtained from Zhejiang TactArtiste Biotechnology Group Co. Ltd, China. Loperamide hydrochloride was purchased from Hangzhou Pharmacy (Xi'an Janssen Pharmaceutical Ltd., Xi'an, China). Rutin, kaempferol, quercetin and nicotiflorin (purity ≥ 98.0 %) were acquired from Sigma-Aldrich, USA. HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany).
The animal study was reviewed and approved by The Ethics Committee of Zhejiang Chinese Medical University, China (JN. No 202110716), and this experiment was conducted according to the European Community guidelines.
A total of 100 male BALB/c mice (seven-week-old), provided by the Shanghai Laboratory Animal Center (Shanghai, China), were housed in a standard rodent cage at a constant temperature (20 ± 2 °C) and humidity (50–60 %) under conventional standard laboratory conditions.
Preparation of C.salicifolius ethyl acetate Extract A 5 kg of fresh leaves of C.salicifolius was sieved (40 mesh screen, aperture 0.425 mm) and crushed at room temperature with constant aeration. Dry material (250 g) was macerated in the extraction kettle, followed by adding 20 times volume of 80% ethanol and boiled at 95 °C for 2 h in water bath. The residues were successively extracted with equal-volume petroleum ether, chloroform and ethyl acetate after decompression concentration to no alcohol taste (Wen et al., 2021). Then the extract of C.salicifolius was obtained by concentrating the ethyl acetate fraction at 60 °C. Subsequently, the extract was filtered and centrifuged (6 000 g for 10 min), supernatant was collected and freeze dried by freeze dryer (Christ Delta LSC, German). Extraction yield (%) = Ws/W0 ×100 (Ws shown the weight of extract after concentration and drying, W0 illustrated the weight of ethyl acetate extract). After the extraction, 32.68 g lyophilized powder of EAECS was obtained.
High Performance Liquid Chromatography (HPLC) Analysis The extract was prepared in a 10 mL volumetric flask, added by methanol to dissolve and dilute the solution to the scale. The sample solution was filtered by 0.45 µm microporous membrane after mixing fully. Main compounds of C. salicifolius extract (0.1g/mL) were analyzed using HPLC system (Agilent 1260). A ZORBAX Extend-C18 liquid chromatography column (250 mm × 4.6 mm, 5 µm) was used for separation and the temperature was maintained at 25 °C. 10 µL of sample was injected into the system with 1 mL/min flow rate. Mobile phase consisted of acetonitrile (A)-0.2 % glacial acetic acid water (B). The linear gradient elution started at 15.5 %–17.5 % (A), changed to 17.5 %–40 % (A) after 20 min, and changed to 40 %–15.5 %(A) after 30min. The extract was monitored by diode array detector at 365 nm. The standard substances of rutin, kaempferol, quercetin and nicotiflorin were used for analysis. By the basis of their peak area, compounds of C.salicifolius extract were quantified with calibration curves of the corresponding standard substance.
Induction of constipation and experimental design The lyophilized powder of C.salicifolius was dissolved in 5 % sterile physiological saline (0.85 % NaCl) into three different concentrations (0.25, 0.5, and 0.75 g/mL of crude drug). Mice were administrated intragastrically with EAECS extract by 10 mL/kg body weight (equivalent to 5, 10, and 15 g/kg). A total of 100 male BALB/c mice were adapt to their new environment for 7 days, divided into two groups for the defecation and intestinal tract assays, respectively. In each test, 50 mice were randomly divided into five groups (n = 10 for each group) and classified into a normal control group (control), loperamide-induced group (Model), 5 g/kg EAECS, 10 g/kg EAECS and 15g/kg EAECS group. Apart from the control group, loperamide hydrochloride (10 mg/kg, 100 mL sterile water) was intragastrically administered daily to the mice in the other four groups for 14 days. After administering, control and constipated mice were fed (basal maintenance fodder: corn, bran, soybean, chicken and fish meal) and drinked freely. The mice in the control group were given a saline solution, while the mice in different doses of EAECS groups were simultaneously administrated with 5, 10 and 15 g/kg EAECS, respectively for 14 consecutive days. Constipation symptoms were confirmed by measuring the number of feces. The body weights and water intake of the mice in all groups were monitored each day throughout the experimental period. On day 14, all groups of mice were fasted with free access to water overnight for the following tests.
Defecation test The defecation assay was conducted and improved according to the method used by Huang et al., 2020. On day 15, 0.2 mL of activated carbon suspension (50 g activated carbon powder dissolved in 10% gum arabic solution) was given for all mice by gavage. Then the mice were immediately placed in separate, clean cages and provided with food and water ad libitum. The time of first black stool defecation was observed and recorded. The feces of each mouse were collected and weighed in individual sterile tubes. The feces were dried in an oven at 65°C for 24 h. The stool water content was calculated as the following formula: stool water content (%) = (wet weight of stool- dry weight of stool) / wet weight of stool ×100 (Huang et al, 2020).
After the defecation test, freshly collected stool samples from each group were immediately frozen in a liquid nitrogen tank and stored in a refrigerator at −80°C for the following assay.
Gastrointestinal transit assay The GI transit test was determined using the method of Huang et al., 2020 with some modifications. On day 15, 0.2 mL of activated carbon meal solution was administered to the mice. After thirty minutes, the mice were euthanized, collected the whole small intestine to measure the length and transit distance. The GI transit rate was calculated using the following equation: Intestinal transit ratio (%) = [the distance traveled by the activated carbon (cm) / the length of the intestine (cm)] × 100 (Qiao et al., 2021).
Analysis of Intestinal Microbiota The Power Soil DNA Isolation Kit (MO BIO, Cat.No.12888, San Diego, United States) was used to extract the microbial genomic DNA from the collected stool samples. DNA purity and concentration were detected by agarose gel electrophoresis. After quantifying DNA, a polymerase chain reaction (PCR) analysis of the V4 variable region of the bacterial 16S rRNA was amplified used specific primer:515F(5′-GTGCCAGCMG CCGCGGTAA-3′); 806R(5′-GGACTACHVGGGTWTCTAAT-3′). The PCR product was confirmed by using 1% agarose gel electrophoresis. The amplified products were purified with Beckman DNA Clean Beads and quantified by the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) (Qiu et al., 2018). Lastly, the PCR purified products were sequenced on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) according to the protocol described (Caparison et al., 2020).
Bioinformatic Analysis Single-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Quality filtering on the raw reads were performed under specific filtering conditions to obtain the high-quality clean reads according to the quality controlled process (Avershina et al., 2013). Sequences analysis was performed by Uparse software (http://drive5.com/uparse/). Sequences with 97 % similarity cutoff were assigned to the same OTUs. OTUs abundance information were normalized using a standard of sequence number corresponding to the sample with the least sequences. Principal Coordinate Analysis (PCoA) was used to analyse the diversity between groups. The alpha diversity was calculated to analyze the diversity of the microbial community by using QIIME (Version 1.7.0). R software (Version 2.15.3) was performed to determine the beta diversity and compare the differences between treatment groups (Bokulich et al., 2013).
Statistical analyses All data were shown as mean ± SD. Statistical analysis was calculated and plotted using GraphPad 9.3 (GraphPad Software, Canada). Differences between groups were analyzed using one-way ANOVA with LSD post hoc test. p < 0.05 was regarded as statistically significant.
Main components in EAECS The extraction yield of EAECS was 62.5 %, after the extraction, 32.68 g lyophilized powder of EAECS was obtained. The HPLC results was represented in Figure 1. The main components of EAECS were rutin, nicotiflorin, quercetin and kaempferol. The instrument precision inspection showed that the standard deviations were 0.46 %, 0.35 %, 0.68 % and 0.68 %, respectively. The data were less than 2%, indicated that the precision of the instrument. The content and retention time of rutin, nicotiflorin, quercetin and kaempferol in EAECS were 68.75, 15.58, 28.65, 15.68 mg/g, 13.26, 15.36, 26.85, 31.86 min respectively (Table 1).
HPLC analysis of EAECS (A) and mixed standard samples (B). The peak identifications are (1) rutin; (2) nicotiflorin; (3) quercetin;(4) kaempferol.
| Component | Linear relationship | Relevance | Content (mg/g) |
|---|---|---|---|
| Rutin | y = 2386.9x + 0.0352 | 0.9993 | 68.75 |
| Nicotiflorin | y = 12654x + 5.5436 | 0.9995 | 15.58 |
| Quercetin | y = 14627x + 0.3275 | 0.9995 | 28.65 |
| Kaempferol | y = 12496x + 8.0263 | 0.9993 | 15.68 |
We identified the rutin (natural flavonol glycoside), mcotifloria (kaempferol-3-O-rutinoside), quercetin (flavonoid monomer compound) and kaempferol (flavonols) which belonged to the major group of flavonoids. Previous reports showed that flavonoids extracted from two Libyan brown algae significantly contributed to the antibacterial properties of pathogenic bacteria (Alghazeer et al., 2017). The flavonoid compounds (rutin and quercetin) from Phoenix loureirii made easily accessible to exert their biological effects in the gastrointestinal tract including antioxidant and bacteriostatic ability (Murugan et al., 2016). Buddleoside as the major flavonoid possessed an important impact on human intestinal microbiota which improved diarrhea and constipation intestinal symptoms (Tao et al., 2016). Studies showed flavonoids (rutin, isofraxidin, scoparone, quercetin and kaempferol) were the most abundant in ethyl acetate extract of C.salicifolius and alkaloid (scopolamine) was accounted the second major component (Wen et al., 2021). The component of C. salicifolius extract might contain flavonoids, alkaloid, polyphenols and polysaccharide (Liu et al., 2013; Pan et al., 2012; Zhang et al., 2014). Some previous studies have suggested these bioactive substances exhibited bacteriostatic ability regulating the abundance and diversity of gut microbiota by producing short chain fatty acids such as acetic, propionic, isobutyric, butyric and valeric acid (Oganezova et al., 2020). Therefore, we designed experiment to reveal the EAECS potential properties on relieving symptoms of intestinal discomfort.
Effect of EAECS on feeding behavior and faeces parameters The feeding behavior of mice and defecation condition was shown in Table 2, there was no significant differences observed in five groups regarding body weight, food and water intake. Compared to the control group, the model group presented significantly lower in the stool pellet number, wet weight and water content of the mice (p < 0.05). For the EAECS treatment groups, fecal excretion and water content of the mice showed a remarkable increase (p < 0.05).
| Category | Group | ||||
|---|---|---|---|---|---|
| Control | Model | 5g/kg EAECS | 10g/kg EAECS | 15g/kg EAECS | |
| Body weight (g) | 35.46 ± 1.56 a | 34.56 ± 1.12 a | 34.48 ± 1.12 a | 34.78 ± 1.43 a | 35.23 ± 0.92 a |
| Food intake (g/d) | 5.42 ± 0.62 a | 5.24 ± 0.17 a | 5.18 ± 0.27 a | 5.36 ± 0.48a | 5.54 ± 0.27 a |
| Water intake (mL/d) | 6.98 ± 0.23 a | 6.87 ± 0.67 a | 7.15 ± 0.27 a | 6.76 ± 0.25 a | 6.82 ± 0.35 a |
| Stool pellet number (ea) | 13.32 ± 2.35 b | 3.92 ± 1.67 a | 13.28 ± 2.85 b | 14.26 ± 3.02 bc | 16.85 ± 2.63 c |
| Stool wet weight (mg) | 132.6 ± 21.5 b | 36.8 ± 10.8 a | 136.8 ± 35.9 b | 158.6 ± 28.9 b | 198.6 ± 46.4 c |
| Stool water content (%) | 62.5 ± 1.5 b | 41.0 ± 2.7 a | 76.6 ± 2.1 d | 67.8 ± 1.2 c | 78.2 ± 2.9 d |
Data show the mean ± SD (n = 10), mean values with different letters are significantly different (p < 0.05).
Effect of EAECS on Time to First Black-Stool Defecation The time to the first black stool defecation was used to indicate the effect of different treatments on constipation in mice. As shown in Figure 2, compared to the model group, no significant differences were observed in EAECS treatments and control group (p > 0.05). The time to the first black stool defecation of mice in the control group was 160 min, which significantly increased to 245.0 min after loperamide induced (p < 0.05). However, the treatments of EAECS to mice were evaluated to reduce the time of first black stool defecation. The 15 g/kg EAECS group showed the shortest time for 151.3 min, which was not significant difference from other EAECS groups (p > 0.05).
Time to the first black-stool defecation. The time referred to the first black stool produced by mice administered. Data represent the mean ± SD, mean counts of ten trials.
Effect of EAECS on the Gastrointestinal Transit Rate The gastrointestinal transit rate indicates the feature of peristalsis and transition during digestion in intestine. The effect of EAECS treatment on the gastrointestinal motility was observed in Figure 3, after administration of loperamide, the gastrointestinal transit rate showed a remarkable decrease (from 55.25 % to 38.02 %), which significantly differed from that of the mice in control and treatment groups (p < 0.05). This data indicated that the constipation model of peristalsis obstruction of small intestine has been successfully established. The highest transit rate of intestinal occurred in the mice treated with 15g/kg EAECS, which significantly increased comparison to the model group (p < 0.05). There were no significant differences showed in control, 5 g/kg and 10 g/kg EAECS group. Based on the time to the first black stool defecation and the gastrointestinal transit rate, 15 g/kg EAECS treatment exhibited best effect, which significantly improved the gastrointestinal transit rate.
Gastrointestinal transit rate of mice. Intestinal transit ratio (%) = [the distance traveled by the activated carbon (cm) / the length of the intestine (cm)] × 100. Data represent the mean ± SD, mean counts of ten trials.
Effect of EAECS treatment on the Alpha and Beta diversities of Microbiota Alpha diversity reflects abundance of the microbial community in the sample, which is used to evaluate the diversity of microbial species including ace, Chao1, PD-whole-tree, observed-species, Simpson and Shannon index. For alpha Diversity in Figure 4, loperamide-induced constipation symptoms, as evidenced by significantly decreased alpha index exception for Simpson index, as compared to the control and EAECS treatment group (p < 0.05). The index exhibited significant increase in the abundance of the community with the higher value. However, Simpson index indicated lower diversity with the high data. For the ace, observed species, simpson index, mice treated with 15 g/kg EAECS exhibited remarkable differences from control and model groups, which significantly improved abundance and diversity of the microbial (p < 0.05).
Alpha diversity indexes analysis of microbial communities. (I): ACE; (II): chao 1; (III): PD-whole-tree; (V): observed-species; (VI): Simpson; (VII): Shannon; A: control group; B: model group; C: 5 g/kg EACES; D: 10 g/kg EACES; E: 15 g/kg EACES. Data represent the mean ± SD, mean counts of tri-trials, mean values with different letters over the bars are significantly different (p < 0.05).
As shown in Figure 5, beta diversity, analyzed by multivariable statistical method PCoA, which was used to evaluate the similarity and difference of microbial evolution in environmental samples. Figure 5 showed a weighted uniFrac matrix of diversities treated with five groups at the genus level, which distinguished differences in species abundance. The distribution of the two principal components represented 78.91 % (PC1) and 13.82 % (PC2) of the accumulative variance contributions, for a total of 92.73 %. The microbial of the mice treated with control, model and EAECS group was separate independently, which indicated more change of structure and abundance of intestinal microbiota.
Principal component analysis of microbial communities in mice. A: control group; B: model group; C: 5 g/kg EACES; D: 10 g/kg EACES; E: 15 g/kg EACES.
Effect of EAECS on the composition of Fecal Microbiota at the Genus Level According to the annotation results of species, the relative abundances of each group at the genus level are shown in Figure 6. A total of 576 bacterial were identified, of which 10 genera were classified to have relative abundances. The relative abundance of Ruminococcus was greatest in the mice of the control group (22.7 %), followed by several genera from Lactobacillus (20.18 %), Lachnospira (16.28 %), and Muribaculace (13.3 %). Bacteroides and Bifidobacterium also accounted for large proportions (6.43 % and 3.48 %, respectively). The model group with loperamide treatment resulted in great change in mice on the relative abundance of microbial community, the proportions of Bacteroide, Muribaculace and Lactobacillus dropped significantly, whereas Ruminococcus obviously increased.
Relative abundances of main genus in the different mice groups. A: control group; B: model group; C: 5 g/kg EACES; D: 10 g/kg EACES; E: 15 g/kg EACES.
The relative abundance of different bacteria was shown in Figure 7. A significant decrease in the relative abundance of Ruminococcus was observed following treatment with EAECS groups compared with the model group. Ruminococcus was usually classified to Firmicutes and Muribaculace was belonged for Bacteroidetes. Firmicutes and Bacteroidetes were the main bacteria in the mucous membrane and the stool respectively. The ratio between Firmicutes and Bacteroidetes increased can lead to fat accumulation and disorder of metabolism such as constipation (Meng et al., 2020). Concentration of SCFAs in the stool of obese and constipation children was lower in comparison to the stool of normal weight children, Firmicutes (Clostridium) predominated in stool microbiota of constipation subjects, while those of Bacteroidetes (Prevotella and Bacteroides) were in minority (Renata et al., 2018).
Changes of relative abundance in genus level of faecal microbiota in different groups of mice. A: model group; B: control group; C: 5 g/kg EACES; D: 10 g/kg EACES; E: 15 g/kg EACES. Data represent the mean ± SD, mean counts of tri-trials.
After the administration of EAECS, the relative abundances of Lactobacillus and Bifidobacterium increased significantly (p < 0.05). Lactobacillus and Bifidobacterium are beneficial to human body, which exhibited important physiological functions such as promoting nutrient absorption and regulating metabolism of host (Naseer et al., 2020). For the Lactobacillus, the relative abundance of 10 g/kg and 15 g/kg EAECS group markedly increased compared with model group. However, group of 10g/kg EAECS compared to both of model and control groups, there was significantly increasing in the relative abundance of Bifidobacterium (p < 0.05).
Constipation is a common gastrointestinal disorder involved with dysphoria and altered bowel movement worldwide and seriously affects human quality of life (Min et al., 2020). Although related medications are effective for treating constipation, most are drug-dependent. Previous studies have suggested that intestinal tract disorder and change was considered as an important factor in constipation. Most modern medical research of constipation has concerned on probiotics, supplemented to relieve symptoms, which reported to be due to their metabolites such as SCFAs and tryptamine (Bharucha et al., 2017). However, there have been less reports focusing on plant extract provided by natural resources in the degree of alleviation of constipation or the resulting changes in the fecal microbiota in mice.
C.salicifolius distributed in the mountain areas, which considered as a unique plant in Eastern China have been applied for drinking tea. The root, stem and leaf of this plant reported for use especially for leaves. The study of C.salicifolius extract showed functional component were extractive from both of alcohol and aqueous solution. Four chemical substances including rutin, nicotiflorin, quercetin and kaempferol indicated that there was difference from aqueous of C.salicifolius reported by previous study (Liu et al., 2013). Remarkablely, these components belonged to favonoid glycosides and widely existed in plants, which was capable for bacteriostasis. The extract of C.salicifolius proved to have a broad inhibitory activity against Escherichia coli and Staphylococcus aureus. Moreover, the aqueous extract could protect mice against mucositis by inhibiting apoptosis and inflammation, the mechanism may be related to the anti-inflammatory and antioxidant effects (Wen et al., 2021). However, bacteriostatic properties of C.salicifolius in the treatment of constipation have not been reported from reference. There were fewer reports explored the effect of intestinal tract revolution mechanism of C.salicifolius extract on constipation.
In this paper, fecal parameters including stool wet weight, content and pellet numbers was used to verify constipation condition induced by loperamide from different treatments. The model group was established successfully accompany with defecate difficulty (Koppen et al., 2018). All dose groups of EAECS exhibited function ability to significantly improve the feces parameters. The time to the first black stool defecation was compared to evaluate bowel movement. Small intestine movement includes three ways: tensional contraction, segmental movement and peristalsis. Changes in intestinal function could promote or inhibit intestinal motility (Min et al., 2020). Gastrointestinal transit rate reflected the effect of treatment on peristalsis during digestion. The GI transit rate of EAECS treatment showed the consistent results of high rate with control compared to model group, 15 g/kg EAECS group markedly reached the top transit rate 76 %. The EAECS treatment indicated the ability to relieve loperamide-induced constipation symptoms in mice.
The species and quantities of intestinal microbiota exhibited differences between constipation patients and healthy people, the abundance of Prevotella decreased significantly compared with the normal subjects (Wang et al., 2021). Previous research suggested that intestinal microbiota in constipation patients compared with normal people showed Enterobacter, Enterococcus, Fusobacterium increased and Bacteroides, Lactobacillus, Bifidobacterium decreased (Kim et al., 2012). In addition, constipation is characterized by prolonged passage of food through the gastrointestinal tract. The colonization of Lactobacillus acidophilus and Bifidobacterium bifidum in mice shortened the length of the change cycle of the transitional actin complex and accelerated the passage time of the small intestine, while Micrococcus luteus and Escherichia coli had the opposite effect (Wang et al., 2021).Therefore, it has been proved that Lactobacillus and Bifidobacterium could produce organic acid, anti-inflammatory cytokines and short chain fatty acid (SCFA) significantly relieved constipation symptoms.
In this paper, the treatment of EAECS groups showed remarkable difference from model group, Ruminococcus abundance of EAECS groups was significantly lower than model group, Ruminococcus affected intestinal metabolism resulted the decreasing of SCFA. Ruminococcus gnavus showed significant negative correlations with acetic, propionic, and butyric acids effected the total and regional body fat (Wei et al., 2018). 10 g/kg EAECS group significantly increased the abundance and diversity of mice induced by loperamide in both Lactobacillus and Bifidobacterium. The main component of EAECS was identified as flavonoids by HPLC. Actually, flavonoids are the most important components in herbal medicine have a wide range of biological activities, especially in antioxidant, antibacterial. After EAECS intervention treatment, the abundance of probiotic anaerobic bacteria contained Lactobacillus, Bifidobacterium significantly increased which produced SCFAs by glycolysis. Adhesion of Bifidobacterium colonized the intestinal tract and regulated levels of SCFAs and improved the intestinal microbiota disorder caused by constipation (López et al., 2012). Moreover, Bifidobacterium induced T cells to differentiate into Treg cells to alleviate gastrointestinal diseases associated with inflammation (Arpaia et al., 2013). The metabolite acetate also stimulated the secretion of 5-HT signal involved in regulating gastrointestinal motility and secretory function. Bifidobacterium effected the secretion of both excitatory and inhibitory transmitters in the enteric nervous system, could relieve constipation by regulating 5-HT signaling system (Bhattarai et al., 2017). This study revealed extract of C.salicifolius had the potential to regulate the intestinal microbial diversity. In the future work, we will isolate and purify EAECS, focus on the mechanisms for EAECS relieving constipation simultaneously.
In conclusion, the main components of EAECS were analyzed and identified for rutin, nicotiflorin, quercetin and kaempferol. We investigated the effects of the administration of EAECS on constipation symptoms based on stool parameters. The treatment of EAECS could significantly reduce the time of first black stool defecation and increase the gastrointestinal transit rate in constipated mice (P < 0.05). Furthermore, the diversity of microbiota in beneficial species were greatly increased by EAECS. The intervention of EAECS on constipated mice effectively regulated intestinal microbiota induced by loperamide and restored their relative abundance. Therefore, the results indicate that ethyl acetate extract of C. salicifolius presents the potential to be developed as a candidate for treating constipation.
Acknowledgements This study was supported by National Natural Science Foundation of China (2019C200302). This project was funded by the Zhejiang Business Collage Foundation of China (KT21001226; KT21001231).
Conflict of interest There are no conflicts of interest to declare.
