In this study, a chemiluminescence (CL) method was developed to determine diphenoxylate in tablets and human plasma. This is the first CL method proposed to determine diphenoxylate. Creating three-dimensional data caused the parallel factor analysis algorithm (PARAFAC) to be used for the first time in CL methods. The method is based on the fact that diphenoxylate enhances the weak CL produced in the reaction of Ru(phen)3 2+ and acidic Ce(IV), and the concentration of Ce(IV) solution has a different effect on the CL response of diphenoxylate and the blank plasma. The calibration curve was linear from 4.0 × 10-8 to 1.6 × 10-6 mol L-1 (R2 = 0.9954), and the detection limit was 1.3 × 10-8 mol L-1 (S/N = 3). The sampling rate was about 30 samples per hour, and the % RSD for 10 repeated measurements of 4 × 10-7 mol L-1 diphenoxylate was 5.4%. The interference effects of some ions, amino acids, and common additives were also investigated. The CL method was successfully used to determine diphenoxylate in tablets, and the results were statistically confirmed by the reference method. The proposed CL method and the PARAFAC algorithm were successfully used to determine the concentration of diphenoxylate in human blood plasma samples.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Chronic constipation has been associated with depression-like behavior. Previous study identified the crucial role of gut microbiota in the development of constipation and depression. Dietary inulin (INU) could regulate gut microbiota. Whether INU treatment could ameliorate constipation induced depression was not clear. For this purpose, male CD-1 mice were administered diphenoxylate (20 mg/kg body weight/day) to induce constipation. We found that INU (10 % in standard diet) alleviated the diphenoxylate-induced constipation, manifested as the increase weight and moisture content of feces. Furthermore, the associated depression and anxiety-like behavior disorders were improved by inhibiting neuro-inflammation and preventing synaptic ultrastructure damage under INU treatment. Moreover, INU pretreatment improved the diphenoxylate-induced gut barrier damage by upregulating tight junction protein expression. INU also reshaped gut microbiota in constipation mice by increasing the relative abundance of Bacteroides and Proteobacteria and downregulating the abundance of Muribacalum and Melaminabacteria. The effects of INU on diphenoxylate-induced depression were abolished by gut microbiota depletion via antibiotic treatment. In addition, INU increased the concentration of short chain fatty acids (SCFAs) in feces contents. Meanwhile, supplementation of SCFAs could also partly improve diphenoxylate-induced depression. In conclusion, INU intake was a potential nutritional intervention strategy to prevent constipation induced depression via microbiota-gut-SCFAs axis.

In clinical trials, rhubarb extract (Rb) was demonstrated to efficiently alleviate constipation. We would like to find out the underlying mechanism of rhubarb relieving constipation. However, there are few studies on the effects of rhubarb on colonic mucus secretion and constipation. The aim of this study was to investigate the effects of rhubarb on colonic mucus secretion and its underlying mechanism. The mice were randomly divided into four groups. Group I was the control group and Group II was the rhubarb control group, with Rb (24 g/kg body weight [b.w.]) administered through intragastric administration for three days. Group III mice were given diphenoxylate (20 mg/kg b.w.) for five days via gavage to induce constipation. Group IV received diphenoxylate lasting five days before undergoing Rb administration for three days. The condition of the colon was evaluated using an endoscope. Particularly, the diameter of blood vessels in the colonic mucosa expanded considerably in constipation mice along with diminishing mucus output, which was in line with the observation via scanning electron microscope (SEM) and transmission electron microscope (TEM). We also performed metagenomic analysis to reveal the microbiome related to mucin gene expression level referring to mucin secretion. In conclusion, Rb relieves constipation by rebuilding mucus homeostasis and regulating the microbiome.

Slow transit constipation (STC) is a prevalent gastrointestinal condition with slow transit, and some probiotics can effectively relieve constipation, but the exact mechanisms have not been fully understood. In this study, we evaluate the impact of Lactiplantibacillus plantarum GUANKE (GUANKE) on diphenoxylate-induced slow transit constipation and speculate on the underlying mechanisms in a mouse model. Administration of L. plantarum GUANKE alleviated constipation indexes, including defecation time, fecal output and water content, and gastrointestinal transit ratio. In addition, GUANKE restored the protein expression of constipation-related intestinal factors (aquaporins (AQPs) and interstitial Cajal cells (ICCs)) in colon tissues measured using immunofluorescence staining; regulated the neurotransmitters and hormones, such as increased levels of 5-hydroxytryptamine, substance P, and motilin; and decreased levels of vasoactive intestinal peptide and nitric oxide in serum, as measured by an ELISA. 16S rRNA and correlation analysis of feces indicated that GUANKE administration effectively reduced constipation-induced Prevotella enrichment and suggested a potential contribution of Prevotella to diphenoxylate-induced STC in mice. GUANKE had no effect on short-chain fatty acids (SCFAs) in cecum content. This study revealed that GUANKE may alleviate constipation in mice through regulating intestinal neurotransmitter and hormone release and altering specific bacterial taxa, rather than by affecting SCFAs and the diversity of microbiota in the gut. Further research is needed to confirm if the findings observed in this study will be consistent in other animal studies or clinical trials.

Objective: To investigate the effects of Mijian Daotong Bowel Suppository (MJDs) on the compound diphenoxylate induced constipation model of male rats and its mechanisms. Methods: Sixty SD male rats were randomly divided into blank group, model group, positive group and MJDs group. The constipation model was established by using compound diphenoxylate gavage. The rats in blank group and model group were treated with saline by enema, the rats in positive group and MJDs group were given Kaisailu and honey decoction laxative suppository by enema, respectively, once a day for 10 days. The body weight, fecal water content, gastric emptying rate (GER) and carbon ink propulsion rate (CIPR) of rats were observed during modeling and administration. The effects of MJDs on the pathological changes of colon tissue in constipation rats were investigated by hematoxylin-eosin (HE) staining. The effect of MJDs on 5-hydroxytryptamine (5-HT) in the colon of constipation rats was investigated by ELISA kit. The effects of MJDs on the expressions of aquaporins 3 (AQP3) and aquaporins 4 (AQP4) in the colon of constipation rats were detected by immunohistochemistry. Results: After 10 days of administration, compared with the blank group, the body weight, fecal water content, carbon ink propulsion rate and colon 5-HT content in the model group were decreased significantly, while the expression levels of AQP3 and AQP4 in the colon were increased significantly (P＜0.05, P＜0.01). Compared with the model group, the fecal water content and colon 5-HT content in the positive group were increased significantly, and the expressions of AQP3 and AQP4 in the colon were decreased significantly. The body weight, fecal water content and colon 5-HT content in the MJDs group were increased significantly, and the expressions of AQP3 and AQP4 was decreased significantly (P＜0.05, P＜0.01). Compared with the positive group, the fecal water content of the MJDs group was decreased significantly, and the expressions of AQP3 and AQP4 in the colon of the MJDs group was decreased significantly (P＜0.05, P＜0.01). Gastric emptying rate was not statistically significant difference between the groups. Conclusion: MJDs has good therapeutic effects on constipation, and its mechanisms may be related to up-regulating the content of 5-HT in the colon and down-regulating the expressions of AQP3 and AQP4 in the colon.
目的: 探索蜜煎导通便栓(MJDs)对复方地芬诺酯诱发雄性大鼠便秘模型的润肠通便作用及机制。方法: 将60只SD雄性大鼠,随机分为空白对照组、模型组、阳性组、MJDs组。采用复方地芬诺酯灌胃法复制便秘模型,空白对照组及模型组灌肠生理盐水,阳性组灌肠给予开塞露,MJDs组灌肠给予蜜煎导通便栓,连续灌肠给药10 d,每天一次。造模及给药期间对大鼠体质量、粪便含水量、胃排空率、碳墨推进率等指标进行观察;采用苏木素-伊红(HE)染色观察MJDs对便秘大鼠结肠组织病理变化的影响;采用ELISA试剂盒观察MJDs对便秘大鼠结肠中5-羟色胺(5-HT)的影响;采用免疫组化法检测MJDs对便秘大鼠结肠中水通道蛋白(AQPs)3、水通道蛋白(AQPs)4表达的影响。结果: 给药10 d后,与空白对照组比较,模型组体重、粪便含水量、碳墨推进率、结肠5-HT含量显著降低,结肠AQP3、AQP4表达量显著增高(P＜0.05,P＜0.01);与模型组比较,阳性组粪便含水量与结肠5-HT含量显著性增加,结肠AQP3、AQP4表达量显著下降,MJDs组体重、粪便含水量与结肠5-HT含量显著升高,AQP3、AQP4表达量显著下降(P＜0.05,P＜0.01);与阳性组比较,MJDs组粪便含水量显著下降,MJDs组结肠AQP3、AQP4表达量显著性下降(P＜0.05,P＜0.01);胃排空率各组间差异无统计学意义。结论: MJDs对便秘具有良好的通便作用,其机制可能与上调结肠5-HT含量,下调结肠AQP3及AQP4表达有关。.

Epidemic increases in opioid deaths prompted policies limiting access to prescription opioids in North America. Consequently, the over-the-counter opioids loperamide (Imodium A-D) and mitragynine, the herbal ingredient in kratom, are increasingly used to avert withdrawal or induce euphoria. Arrhythmia events related to these nonscheduled drugs have not been systematically studied.
In this study, we sought to explore opioid-associated arrhythmia reporting in North America.
The U.S. Food and Drug Administration Adverse Event Reporting System (FAERS), Center for Food Safety and Applied Nutrition Adverse Event Reporting System (CAERS), and Canada Vigilance Adverse Reaction (CVAR) databases were searched (2015-2021). Reports involving nonprescription drugs (loperamide, mitragynine) and diphenoxylate/atropine (Lomotil) were identified. Methadone, a prescription opioid (full agonist), served as a positive control owing to its established arrhythmia risk. Buprenorphine (partial agonist) and naltrexone (pure antagonist), served as negative controls. Reports were classified according to Medical Dictionary for Regulatory Activities terminology. Significant disproportionate reporting required a proportional reporting ratio (PRR) of ≥2, ≥3 cases, and chi-square ≥4. Primary analysis used FAERS data, whereas CAERS and CVAR data were confirmatory.
Methadone was disproportionately associated with ventricular arrhythmia reports (PRR: 6.6; 95% CI: 6.2-7.0; n = 1,163; chi-square = 5,456), including 852 (73%) fatalities. Loperamide was also significantly associated with arrhythmia (PRR: 3.2; 95% CI: 3.0-3.4; n = 1,008; chi-square = 1,537), including 371 (37%) deaths. Mitragynine demonstrated the highest signal (PRR: 8.9; 95% CI: 6.7-11.7; n = 46; chi-square = 315), with 42 (91%) deaths. Buprenorphine, diphenoxylate, and naltrexone were not associated with arrhythmia. Signals were similar in CVAR and CAERS.
The nonprescription drugs loperamide and mitragynine are associated with disproportionate reports of life-threatening ventricular arrhythmia in North America.

BACKGROUND: Slow transit constipation (STC) is a common intestinal disease with increasing incidence. STC results from various factors, such as the enteric nervous system and metabolic changes. As a classical formula of traditional Chinese medicine, Ji-Chuan decoction (JCD) has been extensively and effectively used in STC treatment, yet its pharmacological mechanism remains unclear.
OBJECTIVE: To explore the integrated regulatory pattern of JCD against STC through hyphenated techniques from metabolism, network pharmacology and molecular methods.
METHODS: STC model mice were generated by intragastric administration of compound diphenoxylate (10 mg/kg/d) for 14 d. The STC mice in the low dose of JCD (3.04 g/kg), middle dose of JCD (6.08 g/kg) and high dose of JCD (12.16 g/kg) groups were orally administered JCD solution once a day for 2 wk. The acetylcholine (ACH) level was examined by enzyme-linked immunosorbent assay. The pathological features of colon tissue were observed by hematoxylin and eosin staining. The differentially expressed metabolites and metabolic pathways were tested by nontargeted metabolomics. The main targets and core ingredients of JCD were identified by network pharmacology, and the expression of AKT was confirmed by immunohistochemistry. Finally, the pathways involved in JCD treatment were predicted using a combination of differentially expressed metabolites and targets, and intestinal glial cell apoptosis was demonstrated by immunofluorescence.
RESULTS: JCD significantly promoted intestinal motility, increased the levels of the excitatory neurotransmitter ACH and reduced intestinal inflammation in STC mice. Untargeted metabolomics results showed that JCD significantly restored metabolic dysfunction and significantly affected taurine and hypotaurine metabolism. Network pharmacology and molecular experiments showed that JCD regulates AKT protein expression, and the core component is quercetin. Combined analysis demonstrated that apoptosis may be an important mechanism by which JCD relieves constipation. Further experiments showed that JCD reduced enteric glial cell (EGC) apoptosis.
CONCLUSIONS: This work demonstrated that reducing EGC apoptosis may be the critical mechanism by which JCD treats STC. These findings call for further molecular research to facilitate the clinical application of JCD.

UNASSIGNED: The naturally fermented yak yogurt of pastoralists in the Tibetan Plateau, China, because of its unique geographical environment and the unique lifestyle of Tibetan pastoralists, is very different from other kinds of sour milk, and the microorganisms it contains are special. Lactococcus lactis subsp. lactis HFY14 (LLSL-HFY14) is a new lactic acid bacterium isolated from naturally fermented yak yogurt. The purpose of this study was to study the inhibitory effect of the bacterium on constipation.
UNASSIGNED: Constipation was induced in ICR mice with diphenoxylate, and the constipated mice were treated with LLSL-HFY14. The weight and feces of the mice were visually detected. Colonic tissues were observed on hematoxylin and eosin-stained sections. Serum indices were detected with kits. mRNA expression in the colon was determined by quantitative polymerase chain reaction assay.
UNASSIGNED: Constipation caused weight loss, the number of defecation granules, defecation weight, fecal water content decreased, and the first black stool excretion time increased. LLSL-HFY14 alleviated these symptoms, and the effects were similar to those of lactulose (drug). The pathological examination revealed that constipation caused pathological changes in the colon, and LLSL-HFY14 effectively alleviated the disease. LLSL-HFY14 increased serum levels of motilin, gastrin, endothelin, substance P, acetylcholinesterase, and vasoactive intestinal peptide (VIP) and decreased serum levels of somatostatin in constipated mice. In addition, LLSL-HFY14 upregulated VIP, cAMP, protein kinase A, and aquaporin 3 expression in colonic tissues of constipated mice in a dose-dependent manner.
UNASSIGNED: LLSL-HFY14 inhibited constipation, similar to lactulose, and has the potential to become a biological agent.

Slow transit constipation (STC) has become an epidemic medical problem. There are several kinds of drugs for constipation; however, each drug has its limitations. The gut microbiota has a close relationship with STC. Lactulose is an effective drug for constipation because it is a kind of bulking laxative and microbioecologic, and it relieves the syndromes of STC. We found that the Chinese Herb Solid Drink (CHSD), which contains medicine food homologous materials such as psyllium husk, sweetalmond, semen sesami nigrum, and hemp seed, has a similar effect on relieving constipation as lactulose, although it has different effects on the gut microbiota. We investigated the mechanisms of CHSD in rats with STC, induced by diphenoxylate, via constipation index and enzyme linked immunosorbent assay (ELISA) analyses using serum and 16S rDNA amplicon and gas chromatography-mass spectroscopy (GC-MS). CHSD enhanced the relative abundance of some types of gut microbiota, such as Blautia, Ruminococcus, Roseburia, Coprococcus, Lachnospira, and Phascolarctobacterium, while lactulose enhanced the relative abundance of Blautia, Phascolarctobacterium, Eubacterium, and Akkernansia in diphenoxylate-induced STC rats. Both CHSD and lactulose enhanced the level of short-chain fatty acids in the faeces of rats; however, the composition of those were different between the two drugs. From the perspective of the gut neuroendocrine system, both CHSD and lactulose could elevate neurotransmitters, such as motilin (MTL) and substance P (SP), which promote intestinal peristalsis and reduce the expression of vasoactive intestinal peptide, which inhibits intestinal peristalsis in the serum of STC rats. CHSD could elevate gastrin expression, which also promoted intestinal peristalsis in serum, while lactulose did not have this effect. Our findings suggest that CHSD may be an effective and safe therapeutic choice for STC.