Comparative antioxidant activity and untargeted metabolomic analyses of cherry extracts of two Chinese cherry species based on UPLC-QTOF/MS and machine learning algorithms.

P. pseudocerasus and P. tomentosa are the two native Chinese cherry species of high economic and ornamental worths. Little is known about the metabolic information of P. pseudocerasus and P. tomentosa. Effective means are lacking for distinguishing these two similar species. In this study, the differences in total phenolic content (TPC), total flavonoid content (TFC), and in vitro antioxidant activities in 21 batches of two species of cherries were compared. A comparative UPLC-QTOF/MS-based metabolomics coupled with three machine learning algorithms was established for differentiating the cherry species. The results demonstrated that P. tomentosa had higher TPC and TFC with average content differences of 12.07 times and 39.30 times, respectively, and depicted better antioxidant activity. Total of 104 differential compounds were identified by UPLC-QTOF/MS metabolomics. The major differential compounds were flavonoids, organooxygen compounds, and cinnamic acids and derivatives. Correlation analysis revealed differences in flavonoids content such as procyanidin B1 or isomer and (Epi)catechin. They could be responsible for differences in antioxidant activities between the two species. Among three machine learning algorithms, the prediction accuracy of support vector machine (SVM) was 85.7%, and those of random forest (RF) and back propagation neural network (BPNN) were 100%. BPNN exhibited better classification performance and higher prediction rate for all testing set samples than those of RF. The study herein found that P. tomentosa had higher nutritional value and biological functions, and thus considered for usage in health products. Machine models based on untargeted metabolomics can be effective tools for distinguishing these two species.

Explore the Therapeutic Composition and Mechanism of Schisandra chinensis-Acorus tatarinowii Schott on Alzheimer's Disease by Using an Integrated Approach on Chemical Profile, Network Pharmacology, and UPLC-QTOF/MS-Based Metabolomics Analysis.

Background: Alzheimer's disease places a heavy economic burden to healthcare systems around the world. However, the effective treatments are still lacking. Traditional Chinese medicines (TCM) of Schisandra chinensis and Acorus tatarinowii Schott have the pharmacological effects of sedation and neuroprotection and have been clinically proven to be effective in the treatment of AD. However, their main anti-Alzheimer's compounds and functional mechanisms remain unclear. Purpose: To elucidate the main therapeutic components and possible mechanisms of Sc-At in AD using a comprehensive strategy combining metabolomics and network pharmacology. Methods: First, the UPLC-QTOF/MS method was used to identify the main chemical constituents of Schisandra chinensis and Acorus tatarinowii Schott alcohol extracts in vitro and in vivo. Secondly, the theoretical active ingredients, targets, and pathways of Sc-At for AD treatment were predicted by network pharmacology methods. Finally, plasma metabolomics were detected by UPLC-QTOF/MS to analyze the differential metabolites and metabolic pathways related to Sc-At. Based on the analyses above, the anti-AD mechanism of Sc-At was explored. Results: A total of 95 chemical components were identified in Sc-At extracts in vitro, and 34 prototype drug components were detected in rat plasma; network pharmacology screening identified 14 drug components in line with the principle of Lipinski, of which 10 were present for in vitro drug composition analysis. For these 10 components, 58 AD disease targets were predicted, and 85 AD-related KEGG signaling pathways were enriched. Six core biomarkers of Sc-At (cis-8,11,14,17-eicosatetraenoic acid, prostaglandin H2, sphingosine 1-phosphate, enol-phenylpyruvate, 3-methoxytyrosine, and pristanoyl-CoA) were regulated to a normal state during the treatment of AD. Conclusion: The mechanism of Sc-At for the treatment of AD can be achieved by the effect of the 10 compounds of Sc-At on TNF, MAPK8, MAPK14, PTGS1, and other targets, thereby affecting arachidonic acid metabolism, neurotransmitters, and sphingolipid metabolism.

Develop a stepwise integrated method to screen biomarkers of Baihe-Dihuang Tang on the treatment of depression in rats applying with composition screened, untargeted, and targeted metabolomics analysis.

Baihe-Dihuang Tang is a commonly prescribed remedy for depression. In this study, component screening with untargeted and targeted metabolomics was used to identify potential biomarkers for depression in chronic unpredictable mildly stressed rats. Using this novel identification method, the screening of organic acids, lily saponins, iridoids, and other ingredients formed the basis for subsequent metabolomics research. Baihe-Dihuang Tang supplementation in chronic unpredictable mild-stress-induced depression models, increased their body weight, sucrose preference, brain-derived neurotrophic factor deposition, and spatial exploring. Untargeted metabolomics revealed that Baihe-Dihuang Tang exerts its antidepressant effects by regulating the levels of lipids, organic acids, and its derivatives, and benzenoids in the brain, plasma, and urine of the depressed rats. Moreover, it also modulates the d-glutamine and d-glutamate metabolism and purine metabolism. Targeted metabolomics demonstrated significant reduction in l-glutamate levels in the brains of depressed rats. This could be a potential biomarker for depression. Baihe-Dihuang Tang alleviated depression by regulating the levels of l-glutamate, xanthine, and adenine in the brains of depressed rats. Together, these findings conclusively established the promising therapeutic effect of Baihe-Dihuang Tang on depression and also unraveled the underlying molecular mechanism of its potential antidepressant function.

[Effect of acupuncture on pain-emotion related brain regions in patients with cervical spondylosis of cervical type: a fMRI study].

OBJECTIVE: To observe the changes of functional connectivity of brain pain-emotion regulation region in patients with cervical spondylosis of cervical type by functional magnetic resonance imaging (fMRI). METHODS: Thirty-two subjects were selected. Of them, 16 patients with cervical spondylosis of cervical type were divided into an observation group and 16 healthy subjects into a control group. The patients in the observation group were treated with acupuncture at Tianzhu (BL 10), Jingbailao (EX-HN 15), Jianzhongshu (SI 15) and ashi points for 30 min. The rest-state fMRI data was collected before and after acupuncture in the observation group. The subjects in the control group received no treatment, and the rest-state fMRI data was collected once. The visual analogue scale (VAS) score before and after treatment and the pain catastrophizing scale (PCS) score before treatment in the observation group were recorded. The resting-state brain functional imaging characteristics between the observation group and control group before treatment, between the observation group before and after treatment, were compared. Based on the brain functional connectivity of region of interest (ROI) the changes of functional connectivity in insula and ventral tegmental area (VTA) in emotional regulation brain region were observed, and the correlation between functional connectivity changes and VAS、PCS scores in patients of the observation group was analyzed. RESULTS: In the observation group, the VAS score was (1.94±1.12) after the treatment, which was lower than (5.62±1.20) before treatment (P<0.05). The PCS score before treatment was (19.18±8.42) in the observation group. Compared with the control group, the areas with increased functional connectivity with insula in the observation group before acupuncture included bilateral dorsolateral prefrontal lobe and right middle cingulate gyrus, and the areas with increased functional connectivity with VTA included right central posterior gyrus and right insula. In the observation group, the connectivity coefficient of left insula and left dorsolateral prefrontal lobe (r=0.438, P<0.05), the connectivity coefficient of right insula and right dorsolateral prefrontal lobe (r=0.483, P<0.05) were positively associated with the VAS score. In the observation group, the connectivity coefficient between the right insula and the right middle cingulate gyrus (r=-0.560, P<0.05), the connectivity coefficient between the right VTA and the right insula (r=-0.525, P<0.05) were negatively associated with the PCS score. After acupuncture, the areas with decreased functional connectivity with insula included bilateral posterior central gyrus, right anterior central gyrus, middle cingulate gyrus and left corpus callosum, while the bilateral suboccipital gyrus and left cerebellum showed increased functional connectivity with right insula. The areas with decreased functional connectivity with VTA included bilateral dorsomedial prefrontal cortex, left anterior cingulate gyrus, right middle temporal gyrus and left anterior cingulate gyrus. After acupuncture in the observation group, the functional connectivity of left VTA left dorsomedial prefrontal cortex and left anterior cingulate cortex (r=-0.548, P<0.05), the functional connectivity of right VTA-bilateral dorsomedial prefrontal cortex and left anterior cingulate cortex (r=-0.547, P<0.05) were negatively associated with the PCS score. CONCLUSION: Pain involves the formation and expression of "pain-emotion-cognition". Acupuncture can systematically regulate the brain functional connections between cognitive regions such as dorsal prefrontal lobe and anterior cingulate gyrus and emotional regions such as insula and VTA in patients with cervical spondylosis of cervical type, suggesting that acupuncture has a multi-dimensional and comprehensive regulation effect on pain.

Nuciferine modulates the gut microbiota and prevents obesity in high-fat diet-fed rats.

Gut microbiota dysbiosis has a significant role in the pathogenesis of metabolic diseases, including obesity. Nuciferine (NUC) is a main bioactive component in the lotus leaf that has been used as food in China since ancient times. Here, we examined whether the anti-obesity effects of NUC are related to modulations in the gut microbiota. Using an obese rat model fed a HFD for 8 weeks, we show that NUC supplementation of HFD rats prevents weight gain, reduces fat accumulation, and ameliorates lipid metabolic disorders. Furthermore, 16S rRNA gene sequencing of the fecal microbiota suggested that NUC changed the diversity and composition of the gut microbiota in HFD-fed rats. In particular, NUC decreased the ratio of the phyla Firmicutes/Bacteroidetes, the relative abundance of the LPS-producing genus Desulfovibrio and bacteria involved in lipid metabolism, whereas it increased the relative abundance of SCFA-producing bacteria in HFD-fed rats. Predicted functional analysis of microbial communities showed that NUC modified genes involved in LPS biosynthesis and lipid metabolism. In addition, serum metabolomics analysis revealed that NUC effectively improved HFD-induced disorders of endogenous metabolism, especially lipid metabolism. Notably, NUC promoted SCFA production and enhanced intestinal integrity, leading to lower blood endotoxemia to reduce inflammation in HFD-fed rats. Together, the anti-obesity effects of NUC may be related to modulations in the composition and potential function of gut microbiota, improvement in intestinal barrier integrity and prevention of chronic low-grade inflammation. This research may provide support for the application of NUC in the prevention and treatment of obesity.

Increased BBB permeability contributes to EGCG-caused cognitive function improvement in natural aging rats: pharmacokinetic and distribution analyses.

Previous studies report that (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenolic ingredient in green tea, has high efficacy against Alzheimer's disease (AD) in various in vivo and in vitro models. However, as a water-soluble component, how EGCG exerts its anti-AD effects in the brain was not elucidated. In the present study, we investigated the anti-AD mechanisms of EGCG in natural aging rats with cognitive impairments (CIs) assessed using Morris water maze. The rats were treated with EGCG (100 mg/kg per day, intragastrically) for 4 weeks. The expression of ß-amyloid (Aß1-42) in the brain was detected with immunohistochemical staining. We showed that EGCG administration significantly ameliorated the CI in the aging rats with CI and decreased Aß1-42 plaque formation in their brains. Then we used an efficient ultra-performance liquid chromatography-tandem mass spectrometer method to evaluate EGCG concentrations in rat plasma and tissue distribution. We found that EGCG absorption was significantly increased in the aging with CI group compared with control young rats. After oral administration of EGCG (100 mg), EGCG could not be detected in the brain tissues of control young rats, but it was found in the brain tissue of aging rats with CI. By using Evans Blue assay, transmission electron microscopy, and Western blotting assay, we demonstrated that the permeability of blood-brain barrier (BBB) was significantly increased in aging rats with CI. These results suggest that the permeability change of BBB is the physiological structural basis for EGCG treatment to improve learning and memory, thus providing a solid evidence for EGCG druggability in anti-AD therapeutic field.

Development of a UPLC-MS/MS Method for Simultaneous Determination of Six Flavonoids in Rat Plasma after Administration of Maydis stigma Extract and Its Application to a Comparative Pharmacokinetic Study in Normal and Diabetic Rats.

Maydis stigma is an important medicine herb used in many parts of the world for treatment of diabetes mellitus, which main bioactive ingredients are flavonoids. This paper describes for the first time a study on the comparative pharmacokinetics of six active flavonoid ingredients of Maydis stigma in normal and diabetic rats orally administrated with the decoction. Therefore, an efficient and sensitive ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of six anti-diabetic ingredients (cynaroside, quercetin, luteolin, isorhamnetin, rutin and formononetin) of Maydis stigma in rat plasma has been developed and validated in plasma samples, which showed good linearity over a wide concentration range (r² > 0.99), and gave a lower limit of quantification of 1.0 ng·mL-1 for the analytes. The intra- and interday assay variability was less than 15% for all analytes. The mean extraction recoveries and matrix effect of analytes and IS from rats plasma were all more than 85.0%. The stability results showed the measured concentration for six analytes at three QC levels deviated within 15.0%. The results indicated that significant differences in the pharmacokinetic parameters of the analytes were observed between the two groups of animals, whereby the absorptions of these analytes in the diabetic group were all significantly higher than those in the normal group, which provides an experimental basis for the role of Maydis stigma in anti-diabetic treatment.

Toxicological effects of soil contaminated with spirotetramat to the earthworm Eisenia fetida.

The aim of this study was to evaluate the potential toxicity of spirotetramat to the earthworm Eisenia fetida in a natural soil environment. Many biochemical markers, viz., superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), cellulase, and malondialdehyde (MDA) contents were measured after exposure to 0.25, 1.25, and 2.5mgkg(-1) for 2, 7, 14, 21, and 28days. In addition, the comet assay was performed on earthworm coelomocytes to assess the level of genetic damage. The results demonstrate that the SOD activity and MDA content were significantly stimulated by the highest dose (2.5mgkg(-1)) of spirotetramat for the entire period of exposure. The activities of CAT and POD increased significantly by 2d and 21d, respectively, but the activities of both were significantly inhibited after prolonged exposure (28d). After an initial increase on the 2nd day, the cellulase activity in the high-dose treatment group was significantly inhibited for the entire remaining exposure period. The comet assay results demonstrate that spirotetramat (⩽2.5mgkg(-1)) can induce low and intermediate degrees of DNA damage in earthworm coelomocytes. The results indicate that spirotetramat may pose potential biochemical and genetic toxicity to earthworms (E. fetida), and this information is helpful for understanding the ecological toxicity of spirotetramat on soil invertebrate organisms.

The Maillard reaction of a shrimp by-product protein hydrolysate: chemical changes and inhibiting effects of reactive oxygen species in human HepG2 cells.

Recently, much attention has been given to improving the antioxidant activity of protein hydrolysates via the Maillard reaction, but little is known about the cellular antioxidant activity of Maillard reaction products (MRPs) from protein hydrolysates. We first investigated chemical characterization and the cellular antioxidant activity of MRPs in a shrimp (Litopenaeus vannamei) by-product protein hydrolysate (SBH)-glucose system at 110 °C for up to 10 h of heating. Solutions of SBH and glucose were also heated alone as controls. The Maillard reaction greatly resulted in the increase of hydroxymethylfurfural (HMF) and browning intensity, high molecular weight fraction, and reduction of the total amino acid in SBH with the heating time, which correlated well with the free radical scavenging activity of MRPs. MRPs had stronger inhibiting effects on oxidative stress of human HepG2 cells than the original SBH, and its cellular antioxidant activity strongly correlated with free radical scavenging activity, but less affected by the browning intensity and HMF level. The caramelization of glucose partially affected the HMF level and free radical scavenging activity of MRPs, but it was not related to the cellular antioxidant activity. The cellular antioxidant activity of MRPs for 5 h of heating time appeared to reach a maximum level, which was mainly due to carbonyl ammonia condensation reaction. In conclusion, the Maillard reaction is a potential method to increase the cellular antioxidant activity of a shrimp by-product protein hydrolysate, but the higher HMF levels and the lower amino acid content in MRPs should also be considered.

Determination of monoamine and amino acid neurotransmitters and their metabolites in rat brain samples by UFLC-MS/MS for the study of the sedative-hypnotic effects observed during treatment with S. chinensis.

Schisandra chinensis (Turcz.) Baill. has been used as a sedative and hypnotic agent in traditional Chinese medicine for centuries. The purpose of this study was to reveal the influence of insomnia on the levels of the neurotransmitters: glutamate (Glu), γ-aminobutyric acid (GABA), noradrenaline (NE), dopamine (DA), serotonin (5-HT) and their metabolites (5-HIAA, DOPAC and HVA), and to study the role of S. chinensis in the treatment of insomnia. To achieve this goal, an efficient, sensitive and selective method was developed and validated for the simultaneous determination of these five neurotransmitters and their metabolites in rat brain samples using ultra fast liquid chromatography/tandem mass spectrometry (UFLC-MS/MS). The analysis was performed on a Synergi Fusion-RP 80A ODS column (150mm×2.0mm, 4.0µm) using gradient elution, with the mobile phase consisting of acetonitrile and 0.05% formic acid in water. The method was validated using rat brain homogenate samples and showed a good linearity over a wide concentration range (r(2)>0.99) with a lower limit of quantification (LLOQ) at 4-16ngmL(-1). The intra and inter-day assay variability was less than 15% for all analytes. The results indicated that the condition of insomnia elevated GABA, NE, DA, DOPAC and HVA, and reduced 5-HT, 5-HIAA levels in rat brain. The oral administration of S. chinensis (7.5gkg(-1)day(-1), eight days) influenced insomnia by significantly increasing or reducing the levels of the neurotransmitters parameters mentioned above. These results suggested that S. chinensis could alter the levels of these brain neurotransmitters and their metabolites through its sedative-hypnotic effects.

UFLC-MS/MS method for simultaneous determination of six lignans of Schisandra chinensis (Turcz.) Baill. in normal and insomniac rats brain microdialysates and homogenate samples: towards an in-depth study for its sedative-hypnotic activity.

Schisandra chinensis (Turcz.) Baill., a traditional Chinese medicine, has been clinically used for the treatment of insomnia for centuries. The insomnia mechanism and the possible active ingredients of S. chinensis remain largely unknown. The objective of this study was to develop a method to detect its components which could pass through the blood brain barrier (BBB) by determining the brain microdialysate and brain tissue homogenate samples and then obtain the pharmacokinetic profile in brain for comprehensive understanding of its hypnotic clinical efficacy. Therefore, an efficient, sensitive and selective ultra fast liquid chromatography/tandem mass spectrometry method for the simultaneous determination of six sedative and hypnotic lignans (schisandrin, schisandrol B, schisantherin A, deoxyshisandrin, γ-schisandrin and gomisin N) of Schisandra chinensis (Turcz.) Baill. in rat brain tissue homogenate and brain microdialysates has been developed and validated. The analysis was performed on a Shim-pack XR-ODS column (75 mm × 3.0 mm, 2.2 µm) using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid water. The method was validated in brain homogenate and microdialysate samples, which all showed good linearity over a wide concentration range (r(2)> 0.99), and the obtained lower limit of quantification was 0.1 ng · ml(-1) for the analytes in brain microdialysate samples. The intra- and inter-day assay variability was less than 15% for all analytes. The study proved the six lignans, as sedative and hypnotic ingredients, could pass through the BBB with brain targeting, distributed mainly in the hypothalamus and possessed complete pharmacokinetics process in brain. The results also indicated that significant difference in pharmacokinetic parameters of the analytes was observed between two groups, while absorptions of these analytes in insomniac group were significantly better than those in normal group.

Development of a UFLC-MS/MS method for simultaneous determination of six lignans of Schisandra chinensis (Turcz.) Baill. in rat plasma and its application to a comparative pharmacokinetic study in normal and insomnic rats.

Schisandra chinensis (Turcz.) Baill., a traditional Chinese medicine, has been used for treating insomnia for centuries. This paper was designed to study on the plasma pharmacokinetic for its absorption process, and to compare the pharmacokinetics of its active ingredients in normal and insomnic rats orally administrated with the prescription. Therefore, an efficient, sensitive and selective ultra fast liquid chromatography/tandem mass spectrometry (UFLC-MS/MS) method for the simultaneous determination of six sedative and hypnotic lignans (schisandrin, schisandrol B, schisantherin A, deoxyshisandrin, γ-schisandrin and gomisin N) of Schisandra chinensis (Turcz.) Baill. in rat plasma has been developed and validated. The analysis was performed on a Shim-pack XR-ODS column (75mm×3.0mm, 2.2µm) using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid waterat a flow rate of 0.4ml/min. The detection of the analytes was performed on 4000Q UFLC-MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The method was validated in plasma samples, which showed good linearity over a wide concentration range (r(2)>0.99), and obtained lower limits of quantification were 10, 1.2, 1.2, 1.2, 1.0 and 1.2ngmL(-1) for the analytes. The intra- and inter-day assay variability was less than 15% for all analytes. The mean extraction recoveries of analytes and IS from rats plasma were all more than 85.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in rat plasma. The results indicated that significant difference in pharmacokinetic parameters of the analytes was observed between two groups, while absorptions of these analytes in insomnic group were all significantly higher than those in normal group.