Effects of dietary supplementation with Radix Isatidis polysaccharide on egg quality, immune function, and intestinal health in hens.

This study aimed to investigate the effects of supplementing laying hen diets with Radix Isatidis Polysaccharide (RIPS) on egg quality, immune function, and intestinal health. The research was conducted using 288 Hyland Brown hens, which were randomly assigned to four dietary treatments: control (without RIPS), low dose (200 g/t), medium dose (500 g/t), and high dose (1000 g/t) of RIPS. Each dietary treatment was administered to eight replicates of nine hens for nine weeks. The results revealed that RIPS inclusion in diets significantly improved egg quality parameters such as egg shape index, yolk color, haugh unit, and protein height (P < 0.05). Additionally, RIPS supplementation enhanced immune function as evidenced by an alteration in serum biochemical parameters, an increase in the spleen index, and a decrease in the liver index. Further, an evaluation of intestinal health showed that RIPS fortified the intestinal barrier, thus increasing the population of beneficial intestinal bacteria and reducing the abundance of harmful ones. Such mechanisms promoted intestinal health, digestion, and nutrient absorption, ultimately leading to enhanced egg quality. In conclusion, supplementing laying hen diets with RIPS has been demonstrated to improve egg quality by boosting immunity and optimizing intestinal digestion and absorption.

The Effects of Radix isatidis Raw Material on Egg Quality, Serum Biochemistry, Gut Morphology and Gut Flora.

China produces more than 30 million tons of drug residues every year. Therefore, innovative solutions are needed to mitigate environmental damage. Certain plant compounds boost hens' health and performance. Radix isatidis is promising for layer production. This study elucidates the multidimensional impact of Radix isatidis residual material (RIHR) on laying hens, focusing on the egg quality, intestinal health and the microbial landscape. A total of 288 55-week-old Peking powder laying hens with similar laying rates and body weights were randomly divided into four groups, with eight replicates per group and nine hens per replicate. The groups were divided into a control group, an RIHR low-dose group, a medium-dose group and a high-dose group according to a single-factor, completely randomized design. For the three RIHR treatment groups, the added amounts were 5 kg/t, 10 kg/t and 15 kg/t, respectively. Liquid chromatography- mass spectrometry (LC-MS), molecular docking, fluorescence quantitative PCR and other methods were used. The results showed that three main anti-inflammatory and antiviral compounds were identified in RIHR-indirubin (0.21 µg/g), deoxyvasicinone (0.18 µg/g) and epigoitrin (0.39 µg/g). RIHR significantly increased the eggshell thickness, Haugh unit and protein height (p < 0.05). It also had significant antioxidant and anti-inflammatory effects on ilea and ceca (p < 0.05). The microbial analysis demonstrated that RIHR supplementation led to a significant reduction in foregut Lactobacillus levels (p < 0.05). In the hindgut, a significant increase in pathogenic bacteria was observed (p < 0.05). The study concludes that RIHR's anti-inflammatory compounds may directly act on the intestinal tract to modulate inflammation, highlighting its potential for targeted interventions in poultry health and nutrition.

Pharmacokinetics and biotransformation investigation in beagle dog of active compounds from naoxintong capsule.

Naoxintong Capsule (NXTC), a standardized herbal medicine, has been widely applied in treating cardiovascular and cerebrovascular diseases with remarkable efficacy. However, the efficacy contributing components of NXTC are unclear, and the in vivo absorption and metabolism processes of NXTC remain largely obscured. In this study, using beagle dog as model species, we have identified and tentatively characterized 25 prototype and 15 catabolites of NXTC in beagle dog plasma by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS). We have proposed the in vivo bio-transformation pathways of these absorbed constituents. In addition, for six crucial components, we have developed a quantitative method and conducted plasma pharmacokinetic study of these six components by rapid resolution liquid chromatography tandem triple quadrupole mass spectrometry (RRLC-QQQ-MS/MS). In conclude, our study provided comprehensive insights into the understanding of the plasma absorbed components profiling of NXTC as well as their in vivo transformation behaviors, which would be of great value for identifying efficacy contributing critical components as well as mechanism related investigations of NXTC in the future.

Protective effects of Naoxintong capsule alone and in combination with ticagrelor and atorvastatin in rats with Qi deficiency and blood stasis syndrome.

CONTEXT: Naoxintong Capsule (NXT), a Chinese medicine, has been widely used for the treatment of coronary heart disease (CHD) in clinics. OBJECTIVE: This study evaluated the cardioprotective effects of NXT alone and in combination with ticagrelor (TIC) and atorvastatin (ATO). MATERIALS AND METHODS: Qi deficiency and blood stasis rats were established by 8 weeks high fat diet feeding and 16 days exhaustive swimming and randomly divided into seven groups, that is, NXT (250, 500 and 1000 mg/kg/d), TIC (20 mg/kg/d), ATO (8 mg/kg/d), NXT (500 mg/kg/d)+TIC (20 mg/kg/d) and NXT (500 mg/kg/d)+ATO (8 mg/kg/d) group, with oral administration for 12 weeks. The contents of TC, TG, LDL-C, HDL-C, IL-1ß, IL-6, IL-8, TNF-α, AST, ALT, SOD, MDA, CK-MB, LDH, TXA2, PGI2, IgA, IgG, IgM and C3 in serum were measured. RESULTS: NXT + TIC group was significantly superior to the TIC group in decreasing the levels of TC (4.34 vs. 5.54), TG (3.37 vs. 4.66), LDL-C (1.21 vs. 1.35), LDH (4919.71vs. 5367.19) and elevating SOD level (248.54 vs. 192.04). NXT + ATO group was significantly superior to the ATO group in decreasing the levels of AST (195.931 vs. 241.63), ALT (71.26 vs. 83.16), LDH (4690.05 vs. 5285.82), TXA2 (133.73 vs. 158.67), IgG (8.08 vs. 9.80), C3 (2.03 vs. 2.35) and elevating the levels of HDL-C (1.19 vs. 0.91), SOD (241.91vs. 209.49). CONCLUSIONS: The present findings demonstrate that the combined use of NXT with TIC and ATO had better integrated regulating effects than TIC and ATO, respectively. The mechanism of action requires further research.

Integration of molecular networking and fingerprint analysis for studying constituents in Microctis Folium.

Microctis Folium is the dried leaves of a plant (Microcos paniculata L.) used to improve the digestive system, alleviate diarrhoea, and relieve fever, but information regarding its chemical composition has rarely been reported. The traditional research approach of determining chemical composition has included isolating, purifying, and identifying compounds with high-cost and time-consuming processes. In this study, molecular networking (MN) and fingerprint analysis were integrated as a comprehensive approach to study the chemical composition of Microctis Folium by an ultra fast liquid chromatography-photo diode array detector-triple-time of flight-tandem mass spectrometry (UFLC-DAD-Triple TOF-MS/MS). Large numbers of mass spectrometric data were processed to identify constituents, and the identified compounds and their unknown analogues were comprehensively depicted as visualized figures comprising distinct families by MN. A validated fingerprint methodology was established to quantitatively determine compounds in Microctis Folium. Ultimately, 165 constituents were identified in Microctis Folium for the first time and the identified compounds and approximately five hundred unknown analogues were applied to create visualized figures by MN, indicating compound groups and their chemical structure analogues in Microctis Folium. The validated fingerprint methodology was indicated to be specific, repeatable, precise, and stable and was used to determine 15 batches of samples during three seasons in three districts. Furthermore, seasonal or geographic environmental influences on the chemical profile were estimated by principal coordinate analysis. The results can be used to control the quality of Microctis Folium, observe seasonal or geographic environmental influences on the chemical profiles, and provide a reference for further exploitation of potential active unknown analogues in the future.

Integrated metabolomics and gut microbiome to the effects and mechanisms of naoxintong capsule on type 2 diabetes in rats.

Naoxintong Capsule (NXT) is a Traditional Chinese Medicine formulation which has been widely applied in treating cardiovascular and cerebrovascular diseases. Previous studies also reported the potential effects of NXT against diabetes and certain complications, yet its mechanisms remain largely obscured. Herein, in this study, we investigated the anti-diabetic effects of NXT as well as its potential mechanisms. Type 2 diabetes (T2D) was induced in rats by 10-week high-fat diet in companion with a low-dose streptozotocin injection. NXT was administrated for additional 8 weeks. The results showed that NXT exerted potent efficacy against T2D by alleviating hyperglycemia and hyperlipidemia, ameliorating insulin resistance, mitigating inflammation, relieving hypertension, and reducing myocardial injuries. To investigate its mechanisms, by integrating sequencing of gut microbiota and serum untargeted metabolomics, we showed that NXT could significantly recover the disturbances of gut microbiota and metabolic phenotypes in T2D rats. Several feature pathways, such as arachidonic acid metabolism, fatty acid ß-oxidation and glycerophospholipid metabolism, were identified as the potential mechanisms of NXT in vivo. In summary, our study has comprehensively revealed the anti-diabetic effects of NXT which could be considered as a promising strategy for treating metabolic disorders, T2D and diabetic related complications in clinical practice.

Pharmacodynamic and Metabolomics Studies on the Effect of Kouyanqing Granule in the Treatment of Phenol-Induced Oral Ulcer Worsened by Sleep Deprivation.

Oral ulcers are the most prevalent oral mucosal diseases globally, and no specific treatment schemes are currently available due to the complexity of oral ulcer diseases. Sleep deprivation increases the risk of a deterioration in oral health. Kouyanqing Granule (KYQG) has been used for decades in China to treat inflammatory diseases of the mouth and throat associated with the hyperactivity of fire due to yin deficiency syndrome. However, the mechanisms underlying the effects of KYQG in the treatment of oral ulcers are still unclear. The aims of this study were to investigate whether KYQG treatment could attenuate the symptoms of oral ulcers worsened by sleep deprivation and identify the involved metabolic pathways. First, we conducted chemical profiling of KYQG via UPLC-MS analysis. We then combined pharmacological and metabolomics approaches in a phenol-induced rat model of oral ulcers worsened by sleep deprivation. A total of 79 compounds were initially identified. Our observations showed that KYQG treatment induced a significantly higher healing rate in oral ulcers worsened by sleep deprivation. KYQG significantly reduced the levels of 5-HT and GABA in serum, and only decreased the 5-HT level in brain tissue after phenol injury followed by sleep deprivation. Moreover, KYQG administration significantly suppressed systemic inflammation by inhibiting TNF-α, IL-1ß, IL-6, IL-18, and MCP-1. Immunohistochemical analysis further revealed that KYQG inhibited IL-6 expression in buccal mucosa tissues. KYQG treatment also significantly decreased the serum levels of ACTH, CORT, IgM, and 8-OHdG. Serum metabolomics analysis showed that a total of 30 metabolites showed significant differential abundances under KYQG intervention, while metabolic pathway analysis suggested that the altered metabolites were associated with the dysregulation of eight metabolic pathways. Taken together, our results indicated that KYQG attenuates the symptoms of oral ulcers worsened by sleep deprivation probably through the regulation of the neuroimmunoendocrine system, oxidative stress levels, and tryptophan metabolism. This study also provides a novel approach for addressing the increased health risks resulting from sleep deficiency using an herbal medicine formula.

Simultaneously Quantitative Analysis of Naringin and Its Major Human Gut Microbial Metabolites Naringenin and 3-(4'-Hydroxyphenyl) Propanoic Acid via Stable Isotope Deuterium-Labeling Coupled with RRLC-MS/MS Method.

Widespread in citrus fruits, naringin, a natural 2,3-dihydroflavonoid, is of particular interest to scientists and has a broad range of beneficial bioactivities to health. Orally administered naringin remains in the gut tract for a relatively long time because of its low bioavailability. Under the metabolism mediated by human gut microbiota, naringin could be an active precursor for derived metabolites to play important physiological roles. However, naringin and its metabolites are hard to accurately quantify due to severe endogenic interference. In this study, an analytical rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) method coupled with stable isotope deuterium-labeling is developed and validated to simultaneously quantify naringin as well as its major human gut microbial metabolites naringenin and 3-(4'-hydroxyphenyl) propanoic acid. By eliminating the matrix interferences, this strategy not only confirms naringenin and 3-(4'-hydroxyphenyl) propanoic acid as the predominant metabolites which contribute to the pharmacological effects of naringin but also provides a suitable choice for other flavonoid pharmacokinetics study.

Naoxintong Capsule Inhibits the Development of Cardiovascular Pathological Changes in Bama Minipig Through Improving Gut Microbiota.

Naoxintong capsule (NXT), a Chinese medicine, has performed excellent effects on the prevention and treatment against cardiovascular diseases. NXT is a fine powder mixture without any herb extraction, and there must be lots of ingredients hard to be absorbed. However, little is known about the correlation between the NXT's cardioprotective effects and gut microbiota. Herein, we report the effect of NXT on the development of cardiovascular diseases and clarify the correlation between NXT's cardioprotective effects and gut microbiota. In the current study, minipigs were selected and fed with high-fat diet and NXT daily for successive 8 months. During the process, up to 18 biomedical parameters were monthly determined to observe the dynamic changes after NXT treatment. At the end of experimental process, pathological examinations of heart, coronary artery, carotid artery, thoracic aorta, and abdominal aorta were conducted by HE staining and 16SrDNA sequencing, and analyzing of gut microbiota were conducted. Our results showed that NXT's effects against cardiovascular diseases were through regulating blood lipid profiles, inhibiting vascular inflammation, enhancing antioxidant capacity, and alleviating myocardial injury, without damages on liver and kidney particularly. Concurrently, we also found that long-term administration of NXT increased the diversity of gut microbiota, influenced the microbiome structure and composition stably, and revered the increase of the ratio of the Firmicutes to Bacteroidetes (F/B ratio) in relative abundance. Specifically, our results revealed some key bacterium of Caproiciproducens (enhanced), Sutterella (enhanced), Erysipelotrichaceae (enhanced), and Romboutsia (decreased) that were closely involved in NXT's effects. Taken together, our study demonstrates that NXT can inhibit the development of cardiovascular diseases by ameliorating high-fat diet-induced metabolic disorders and partly through improving gut microbiota.

Identification of prototype compounds and derived metabolites of naoxintong capsule in beagle dog urine and feces by UFLC-Q-TOF-MS/MS.

Naoxintong Capsule (NXTC) is a well-known Traditional Chinese Medicine (TCM) medication that has been widely employed in the treatment of cardiovascular and cerebrovascular diseases. Although its chemical constituents had been characterized, the in vivo biotransformation of those components remain obscured. In this study, by applying the ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS), we have investigated the in vivo metabolism of NXTC in beagle dog urine and feces. After a single dose of oral administration, a total of 36 prototype compounds and 52 metabolites of NXTC were identified or tentatively characterized in beagle dog urine and feces. We have also proposed the in vivo transformation pathways of such metabolites including phase I (reduction, oxidation, hydroxylation, demethylation) and phase II reactions (glucuronidation, sulfation, methylation). For the first time, our results have unsealed the in vivo metabolic profiles of chemical components of NXTC in beagle dogs and added novel knowledge into the understanding of efficacy contributing compounds of NXTC that deserves further investigation.

Rotundic Acid Protects against Metabolic Disturbance and Improves Gut Microbiota in Type 2 Diabetes Rats.

Rotundic acid (RA) is a major triterpene constituent in the barks of Ilex rotunda Thunb, which have been widely used to make herbal tea for health care in southern China. RA has a variety of bioactivities such as anti-inflammation and lipid-lowering effect. However, little is known about the effects and mechanisms of RA on metabolic disturbance in type 2 diabetes (T2D) and its effect on gut microbiota. A T2D rat model induced by high fat diet (HFD) feeding and low-dose streptozotocin (STZ) injection was employed and RA showed multipronged effects on T2D and its complications, including improving glucolipid metabolism, lowering blood pressure, protecting against cardiovascular and hepatorenal injuries, and alleviating oxidative stress and inflammation. Furthermore, 16s rRNA gene sequencing was carried out on an Illumina HiSeq 2500 platform and RA treatment could restore the gut microbial dysbiosis in T2D rats to a certain extent. RA treatment significantly enhanced the richness and diversity of gut microbiota. At the genus level, beneficial or commensal bacteria Prevotella, Ruminococcus, Leuconostoc and Streptococcus were significantly increased by RA treatment, while RA-treated rats had a lower abundance of opportunistic pathogen Klebsiella and Proteus. Spearman's correlation analysis showed that the abundances of these bacteria were strongly correlated with various biochemical parameters, suggesting that the improvement of gut microbiota might help to prevent or attenuate T2D and its complication. In conclusion, our findings support RA as a nutraceutical agent or plant foods rich in this compound might be helpful for the alleviation of T2D and its complications through improving gut microbiota.

Identification of naringin metabolites mediated by human intestinal microbes with stable isotope-labeling method and UFLC-Q-TOF-MS/MS.

Widely presented in medicinal plants, naringin is one of the major flavanones with various pharmaceutical bioactivities. After oral administration, naringin predominantly undergoes metabolisms mediated by liver cytochrome P450 and gut microbes, while its human microbes-mediated metabolic profiling is still largely obscure due to the endogenous interferences, which makes it extremely difficult to analyze metabolites precisely. In this study, we aim of systematically investigating the biotransformation of naringin mediated by human intestinal microbes through applying stable isotope-labeling method. [2',3',5',6'-D4]naringin was synthesized and incubated anaerobically with human gut microbes. A total of 13 microbial metabolites were detected and identified by UFLC-Q-TOF-MS/MS, among which 5 were reported for the first time. Furthermore, the proposed metabolic pathway revealed that naringin went through extensive phase I metabolism in human intestinal microbes. Of note, diverse metabolic profiles of naringin among human participants were obtained, which could be attributed to the distinct gut microbiota compositions of individuals.

Simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma by RRLC-MS/MS and its application to a pharmacokinetic drug interaction study.

A rapid resolution liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma. Chromatographic separation of analytes and internal standard (fluvastatin for rosuvastatin, while isoquercitrin for naringin and naringenin) was performed on Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 µm) using gradient elution with a mobile phase of methanol and water, both with 0.1% formic acid (v/v). The detection was operated in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 579.1→270.8 for naringin, m/z 270.9→150.7 for naringenin, m/z 463.1→299.8 for isoquercitrin in negative ionization mode, and m/z 482.2→258.1 for rosuvastatin, m/z 412.1→224.1 for fluvastatin in positive ionization mode. Polarity switch (negative-positive-negative ionization mode) was performed in a total runtime of 5.0 min. The method was validated over a concentration range of 10-2,000 ng/mL for the above three analytes. The intra-day and inter-day precisions and accuracies of the quality control samples at low, medium and high concentration levels exhibited relative standard deviations <10% and the accuracy values ranged from -7.2% to 8.4%. The proposed method was successfully applied to the pharmacokinetic drug interaction study of rosuvastatin combined with naringin in rats.

An experimental model for hypertensive crises emergencies: Long-term high-fat diet followed by acute vasoconstriction stress on spontaneously hypertensive rats.

Currently, the prevention and treatment of hypertensive crises especially when it occurs with serious adverse outcomes have led to worldwide controversy. Despite of clinical possibilities of multiple agents, clinical failures still occur frequently. Therefore, early evaluations and observations of different therapies on appropriate animals should be emphasized. In the present study, an animal model for hypertensive crises emergencies was firstly established and experimentally testified. Five-month-male spontaneously hypertensive rat was consecutively fed with 60%-Kcal fat diet for four, six, and eight weeks with body weight and blood pressure monitored every two weeks, and then followed by an acute vasoconstriction stress of 5-min ice-bath treatment in the 4-h time interval of two adrenaline injections (0.8 mg/kg). Forty-four biochemical parameters were detected, covering hepatic and renal function, blood glucose and lipid levels, myocardial enzymes and energy metabolisms, blood coagulative and anti-coagulative system, oxidative stress and anti-inflammatory cytokine, blood viscosity, and RAAS system. Six tissues including heart, brain, liver, kidney, coronary arteries, and mesenteries were removed for pathological observations with hematoxylin-eosin staining. As a result, multi-organ dysfunctions in the heart, brain, liver, kidney, vascular endothelium, and blood system were testified in the modeling rats at weeks 6 and 8. In conclusion, severe consequences of this animal model were highly similar to those in hypertensive crises emergencies, which could be further utilized in the early intervention of hypertensive crises emergencies including the possible risk factors control and efficient therapies assessment. Impact statement In the late 90s, numerous reports predicted that 1-2% of hypertensive individuals would undergo hypertensive crises (HPC) and figures reached as high as 7% when no antihypertensive therapies were administrated. Currently, clinical failures appear frequently due to the improper or excessive medication regimen instead of the illness itself. Therefore, early evaluations and observations of HPC on appropriate animal models ahead of patients should be discussed and emphasized more widely. In the present study, an appropriate animal model for HPC emergencies was firstly established, in which the consequences of long-term high-fat diet feeding followed by an acute vasoconstriction stress on the spontaneously hypertensive rats were experimentally testified. The proposed model would have a wide application prospects in early intervention of HPC emergencies including the controls of possible risk factors and assessments of efficient therapies.

Urinary metabolite profiling of flavonoids in Chinese volunteers after consumption of orange juice by UFLC-Q-TOF-MS/MS.

The metabolism of flavonoids derived from orange juice in Chinese volunteers has not been well investigated. With the ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS) system, orange juice-derived flavonoids, as well as metabolites contained in urine collected from healthy Chinese volunteers after consumption of 250mL orange juice, were systematically identified and quantified. Finally, a total of 9 flavonoids and 30 metabolites were detected. Obtained results revealed that flavonoids derived from orange juice underwent extensive phase II metabolism in human, mainly comprising glucuronidation and sulfation. The overall recovery of the primary flavonoid aglycones, i.e., naringenin and hesperetin, were both approximately equivalent 22% of intake, primarily occurred in 4-12h post consumption. Meanwhile, additional 35 phenolic catabolites were identified in urine collected post consumption. However, it is difficult to determine the exact amounts of phenolic catabolites derived from specific flavonoid due to the interference of diets and other flavonoids. This work would be valuable for the clarification of metabolic profiles for flavonoids in Chinese population.