Identifying the active compounds and mechanism of action of TongFu XieXia Decoction for treating intestinal obstruction using network pharmacology combined with ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry.

RATIONALE: TongFu XieXia Decoction (TFXXD), a formulation rooted in traditional Chinese medicine and optimized through clinical practice, serves as an advanced version of the classic Da Cheng Qi decoction used for treating intestinal obstruction (IO), demonstrating significant therapeutic efficacy. However, due to the intricate nature of herbal compositions, the principal constituents and potential mechanisms of TFXXD have yet to be clarified. Accordingly, this study seeks to identify the active compounds and molecular targets of TFXXD, as well as to elucidate its anti-IO mechanisms. METHODS: Qualitative identification of the principal constituents of TFXXD was accomplished using ultra-high preformance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS/MS) analysis. PharmMapper facilitated the prediction of potential molecular targets, whereas protein-protein interaction analysis was conducted using STRING 11.0. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the Metascape database. A "compounds-target-pathway" network was meticulously constructed within Cytoscape 3.8.2. Finally, molecular docking studies were performed to investigate the interactions between the core target and the crucial compound. RESULTS: UPLC-Q-Orbitrap-MS/MS analysis identified 65 components with high precision and sensitivity. Furthermore, 64 potential targets were identified as integral to TFXXD bioactivity in IO treatment. Gene Ontology enrichment analysis revealed 995 distinct biological functions, while the Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 143 intricate signaling pathways. CONCLUSION: Molecular docking studies substantiated the substantial affinity between the TFXXD bioactive constituents and their corresponding targets in the context of IO. TFXXD exerts its therapeutic efficacy in IO through a multifaceted interplay between multiple compounds, targets, and pathways. The integration of network pharmacology with UPLC-Q-Orbitrap-MS/MS has emerged as a promising strategy to unravel the intricate web of molecular interactions underlying herbal medicine. However, it is imperative to emphasize the necessity for further in vivo and in vitro experiments.

Uncovering the effective substances and mechanism of Ipomoea pes-caprae against rheumatoid arthritis using liquid chromatography-mass spectrometry and targeted network pharmacology.

Ipomoea pes-caprae (L.) R. Br (Convolvulaceae) is a commonly used marine traditional Chinese medicine in the southern coastal areas of China. It has been widely used to treat rheumatoid arthritis, but its effective substances and anti-rheumatoid arthritis mechanism remain ambiguous. Hence, in this study, the chemical profile and absorbed ingredients of Ipomoea pes-caprae were elucidated by ultra-performance liquid chromatography-mass spectrometry. Moreover, targeted network pharmacology was used to clarify the mechanism of action of Ipomoea pes-caprae in treating rheumatoid arthritis. Finally, 23 compounds were identified in the aqueous extracts of Ipomoea pes-caprae and 12 absorbed ingredients were detected in rats' plasma. These 12 absorbed ingredients might be the essential effective substances of Ipomoea pes-caprae. The tissue distributions of 3 absorbed ingredients in rats were successfully analyzed. The targeted network pharmacological analysis results indicated that the regulation of inflammatory reaction, immune response, cell proliferation, and apoptosis were the critical mechanism of Ipomoea pes-caprae against rheumatoid arthritis. This study successfully clarified the effective substances and potential mechanisms of Ipomoea pes-caprae in treating rheumatoid arthritis. The results of this research could provide a valuable reference for further scientific research and clinical application.

Network Pharmacology Combined with Metabolomics Approach to Investigate the Toxicity Mechanism of Paclobutrazol.

Paclobutrazol (PBZ) is a commonly used plant growth regulator (PGR) with good antibacterial activity. It has widespread applications in agricultural production. However, there is limited research reported on the potential risks of human health resulting from PBZ residues. In this study, using Sprague-Dawley rats, we carried out a systematic study on the hepatotoxicity and nephrotoxicity of PBZ in different doses (0.2, 0.5, and 1.0 g/kg). The metabolic profiles and network pharmacology were combined to construct a PBZ-endogenous substances-gene-hepatorenal diseases network to elucidate the underlying mechanism of PBZ's hepatorenal toxicity. At first, metabolomics analysis was done to investigate the metabolites and the related metabolic pathways associated with PBZ. Secondly, the network pharmacology approach was used in further exploration of the toxic targets. Additionally, molecular docking was carried out to investigate the interactions between PBZ and potential targets. The results indicated that PBZ showed obvious toxicity towards the liver and kidney of rats. The metabolomics analysis showed that PBZ mainly affected 4 metabolic pathways, including tryptophan metabolism, arachidonic acid metabolism, linoleic acid metabolism, and purine metabolism. Network pharmacology and molecular docking revealed that CYP1A2, CYP2A6, CYP2E1, MAOA, PLA2G2A, PTGS1, and XDH were critical targets for PBZ hepatorenal toxicity. This preliminary study revealed PBZ's hepatorenal toxicity and provided a theoretical basis for the rational and safe use of PBZ. Furthermore, it provided possible intervention targets for further research on how to avoid or reduce the damage caused by pesticides to the human body.

The chemical profile of Fubai Chrysanthemum (Fubaiju) and its mechanism in preventing cataract based on ultrahigh-performance liquid chromatography coupled with mass spectrometry and network pharmacology.

Chrysanthemum is a kind of herb that can be used for both medicine and food. Although it has been shown to affect the redox damage of the lens, but the mechanism of action has not been systematically studied. This study identified the chemical profile of Fubai Chrysanthemum. Meanwhile, network pharmacology and the enrichment of the Kyoto Encyclopedia of Genes and Genomes pathway were combined to investigate the substance basis of Fubai Chrysanthemum for preventing cataract. The aqueous extracts of Fubaiju mainly contained 39 compounds. Compared with Gongju, Jinsiju, and wild chrysanthemum, Fubai Chrysanthemum showed a higher scavenging rate of 1,1-diphenyl-2-picrylhydrazyl free radicals and a higher content of total flavonoid. Fourteen chemical differences in four kinds of chrysanthemum were found based on principal component difference analysis. Pathway enrichment analysis showed that the main mechanisms of Fubai Chrysanthemum for preventing cataract were affecting the oxidative stress process and regulating cell growth and metabolism. Eventually, 11 key targets of Fubai Chrysanthemum for cataract prevention were identified. The strategy provided a better understanding of the chemical profile of Fubai Chrysanthemum and elucidated that its higher flavonoid content plays an important role in preventing cataract through antioxidant action and regulating cell growth.

Multiresidue screening of pesticides in Panax Ginseng C. A. Meyer by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

In this study, an efficient screening method based on a modified quick, easy, cheap, effective, rugged, and safe extraction method combined with ultra-high-performance liquid chromatography coupled to tandem quadrupole time-of-flight mass spectrometry was established for the determination of 90 pesticides residues in Panax Ginseng. The accuracy of the method was then verified by analyzing the false positive rate and the screening detection limit in Ginseng. The results revealed that the screening detection limit of 33 of 90 pesticide residues were 0.01 mg·kg-1 , 22 species were 0.05 mg·kg-1 , 11 species were 0.10 mg·kg-1 , 8 species were 0.20 mg·kg-1 , and another 16 species were greater than 0.20 mg·kg-1 . A total of 73 pesticides were ultimately suitable to be practically applied for rapid analysis of pesticide residues in Ginseng. Finally, the established method was used to analyze the pesticide residues in 35 Ginseng samples available on the market. And the residual of dimethomorph, azoxystrobin, tebuconazole, and pyraclostrobin was relatively severe in Ginseng samples. This work expanded the range of pesticides detected and provided a rapid, effective method for pesticides screening in Ginseng.

Pharmacokinetics and tissue distribution study of 18 bioactive components in healthy and chronic heart failure rats after oral administration of Qi-Shen-Ke-Li formula using ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry.

RATIONALE: Qi-Shen-Ke-Li (QSKL) is a traditional Chinese formula used in clinical practice to treat chronic heart failure (CHF) in humans. To rationalize the use of this formula in clinical practice, the pharmacokinetics and tissue distribution in rats after oral administration of QSKL were investigated using ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/TQ-MS). METHODS: The CHF model was induced by intraperitoneal injection of isoprenaline (ISO; also known as isoproterenol) and evaluated by HE staining and brain natriuretic peptide (BNP) measurement. The UHPLC/TQ-MS method was then applied to determine the concentrations of 18 bioactive components in rat plasma and tissues of heathy and CHF rats after oral administration of QSKL. This was followed by investigating the pharmacokinetics and tissue distribution profiles of these bioactive compounds in the heathy and CHF rats. RESULTS: The pharmacokinetics results showed that the duration time of two compounds was prolonged, the absorption rate of four compounds was accelerated, and the bioavailability of four compounds was increased in the CHF rats compared with the healthy rats. Meanwhile, the tissue distribution results showed that the QSKL formula could be distributed rapidly and widely in different rat tissues. The bioavailability of eight compounds in the liver was enhanced in CHF rats. This suggested that the drug/toxic effects should be considered in clinical practice, as drug-drug interactions might occur in liver metabolism during the drug combination. CONCLUSIONS: The pharmacokinetic profiles and tissue distribution of 18 bioactive compounds in QSKL are altered by the CHF status. This study provides insight for better clinical applications of this formula in the future and lays the foundation for the development of a new drug for chronic heart failure based on the QSKL formula.

Mass spectrometry-based urinary metabolomics for exploring the treatment effects of Radix ginseng-Schisandra chinensis herb pair on Alzheimer's disease in rats.

Herb pairs are the unique combinations of two relatively fixed herbs, intrinsically convey the basic idea of traditional Chinese medicine prescriptions. The compatibility of Radix ginseng and Schisandra chinensis has been used in traditional Chinese medicine for treating Alzheimer's disease for many years. However, there are few studies on Radix ginseng-Schisandra chinensis herb pair, and the underlying action mechanism is still unclear. In this study, the mechanism of Radix ginseng-Schisandra chinensis herb pair on Alzheimer's disease was investigated by using the mass spectrometry-based urinary metabolomics method. Sixteen urinary endogenous metabolites were identified as potential biomarkers. Meanwhile, 10 biomarkers were quantified with tandem mass spectrometry. The study result showed that the brain pathologic symptoms of model rats were improved and the potential biomarkers were adjusted backward significantly after the herb pair administration. The metabolic pathways linked to the herb pair-regulated endogenous biomarkers included phenylalanine and tyrosine metabolism, tryptophan metabolism, purine metabolism, and so on. The above metabolic pathways reflected that Radix ginseng-Schisandra chinensis herb pair mainly regulates abnormal energy metabolism, reduces inflammation, and regulates gut microbiota and neurotransmitters in the treatment of Alzheimer's disease.

Comprehensive physiopathology and serum metabolomics for the evaluation of the influence mechanism of qi deficiency on xenograft mouse models of liver cancer.

Traditional Chinese medicine believes that qi deficiency is important pathogenesis and syndrome of liver cancer and thus is crucial in related research. However, the effect of qi deficiency on the occurrence and development of liver cancer is still unclear. This study aimed to establish a liver cancer model of qi deficiency through the swimming exhaustion and xenograft of human hepatoma HepG2 cells. The effects of qi deficiency on the occurrence and development of liver cancer were investigated by analyzing tumor development, blood routine, histopathology, and serum metabolomics. Results showed that qi deficiency greatly affected the physiology and tumor growth of xenograft mice. Eight potential biomarkers were identified by metabolomics based on ultra-high performance liquid chromatography and tandem quadrupole time-of-flight mass spectrometry. Their main pathways were arachidonic acid metabolism, phenylalanine metabolism, purine metabolism, glycerolipid metabolism, steroid biosynthesis, sphingomyelin metabolism, and fatty acid metabolism pathway. Finally, the effects of qi deficiency on the occurrence and development of liver cancer were comprehensively analyzed, and the mechanism of this process was preliminarily clarified.

Poria cocos could ameliorate cognitive dysfunction in APP/PS1 mice by restoring imbalance of Aß production and clearance and gut microbiota dysbiosis.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Amyloid beta-protein (Aß) plaques, which are the hallmark of AD, are formed from the imbalance of Aß production and clearance accompanied by neuroinflammation, gut dysbiosis, and metabolite dysfunction. All of these processes give rise to neurochemical deficiencies and synaptic dysfunction, which ultimately contribute to recognition dysfunction. Poria cocos (PC), which contains multiple active ingredients, plays a significant role in the treatment of multiple-pathogenesis senile diseases such as AD. Nevertheless, there are only very few investigations on the intricate action mechanism of PC for the treatment of AD. In this study, we evaluate the multi-target cure effect of PC on APP/PS1 mice by behavioral, immunohistochemical (IHC), targeted metabolomics, and 16S rRNA sequencing experiments. Mice treated with PC showed significant improvements in cognitive function as evaluated by the behavioral experiment. IHC revealed that PC treatment relieved Aß deposition by reducing the formation of Aß and increasing its clearance. Moreover, PC treatment improved gut dysbiosis, which reversed the metabolite dysfunction of bile acid. These findings reveal that PC is a promising therapeutic agent, which might ameliorate the cognitive function of AD by restoring the imbalance of Aß production and clearance and gut microbiota dysbiosis.

The effects and mechanisms of aloe-emodin on reversing adriamycin-induced resistance of MCF-7/ADR cells.

Multidrug resistance (MDR) is one of the major obstacles for clinical effective chemotherapy. In this study, the effects and possible mechanisms of aloe-emodin (AE) were investigated on reversing the adriamycin (ADR)-induced resistance of MCF-7/ADR cells. AE could significantly reverse the ADR resistance in MCF-7/ADR cells. The combination of AE (20 µM) and ADR had no effect on the P-glycoprotein (P-gp) level, but notably promoted the accumulation of ADR in drug-resistant cells. The efflux function of P-gp required ATP, but AE reduced the intracellular ATP level. AE played a reversal role might through inhibiting the efflux function of P-gp. The research result of energy metabolism pathways indicated that combination of AE and ADR could inhibit glycolysis, tricarboxylic acid (TCA) cycle, glutamine metabolism, and related amino acid synthesis pathways. Moreover, we found AE not only reversed ADR-induced resistant but also induced autophagy as a defense mechanism. In addition, the combination of AE and ADR arrested G2/M cell cycle and induced apoptosis through DNA damage, ROS generation, caspase-3 activation. Our study indicated that AE could be a potential reversal agent to resensitize ADR resistant in tumor chemotherapy and inhibiting autophagy might be an effective strategy to further enhance the reversal activity of AE.

Comparative pharmacokinetics of Ding-Zhi-Xiao-Wan preparation and its single herbs in rats by using a putative multiple-reaction monitoring UPLC-MS/MS method.

INTRODUCTION: The formula of Chinese medicine, Ding-Zhi-Xiao-Wan (DZXW), has the distinct feature of compatibility therapy, which is attributed to the interactions of multi-herbs. However, the quantification problem caused by the absence of pure reference standards is a bottleneck to clarify the compatibility advantages from the perspective of pharmacokinetics (PKs). OBJECTIVE: This study aimed to develop a putative multiple-reaction monitor (PMRM) strategy for exploring the comparative PKs of DZXW and its single herbs. METHODS: First, precursor ion and tandem mass spectrometry (MS/MS) chromatograms were obtained via ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight MS (UHPLC-Q-TOF-MS) under different collision energy (CE) values. Then, the two most abundance ions in the MS/MS chromatograms were chosen as product ions, and CE values were selected according to the abundance of the product ion peaks. Next, a PMRM strategy consisting of optimal MRM parameters was constructed. Finally, the established PMRM parameters were imported to UHPLC coupled with triple quadrupole MS (UHPLC-TQ-MS) for quantification. RESULTS: The strategy was exemplified by the comparative PK study of DZXW and its single herbs. This strategy could extend the PK scopes of multi-components. The quantitative results displayed substantial variations in PK parameters between DZXW and its single herbs. CONCLUSION: The PK parameters indicated that the DZXW formula could increase the exposure levels of most ingredients and reduce the maximum concentration (Cmax ) of Radix Polygala, indicating that herb compatibility could produce synergistic effects and diminish possible toxic effects. This study provides a viable orientation for the compatibility investigation of traditional Chinese medicine.

Scale-Up Preparation of Crocins I and II from Gardeniajasminoides by a Two-Step Chromatographic Approach and Their Inhibitory Activity Against ATP Citrate Lyase.

Crocins are highly valuable natural compounds for treating human disorders, and they are also high-end spices and colorants in the food industry. Due to the limitation of obtaining this type of highly polar compound, the commercial prices of crocins I and II are expensive. In this study, macroporous resin column chromatography combined with high-speed counter-current chromatography (HSCCC) was used to purify crocins I and II from natural sources. With only two chromatographic steps, both compounds were simultaneously isolated from the dry fruit of Gardenia jasminoides, which is a cheap herbal medicine distributed in a number of countries. In an effort to shorten the isolation time and reduce solvent usage, forward and reverse rotations were successively utilized in the HSCCC isolation procedure. Crocins I and II were simultaneously obtained from a herbal resource with high recoveries of 0.5% and 0.1%, respectively, and high purities of 98.7% and 99.1%, respectively, by HPLC analysis. The optimized preparation method was proven to be highly efficient, convenient, and cost-effective. Crocins I and II exhibited inhibitory activity against ATP citrate lyase, and their IC50 values were determined to be 36.3 ± 6.24 and 29.7 ± 7.41 µM, respectively.

Self-Protecting Biomimetic Nanozyme for Selective and Synergistic Clearance of Peripheral Amyloid-ß in an Alzheimer's Disease Model.

Clearance of peripheral amyloid-ß (Aß) has been demonstrated to be promising for overcoming the blood-brain barrier (BBB) hurdle to eliminate brain-derived Aß associated with Alzheimer's disease (AD). Even so, current developed therapeutic assays for clearance of peripheral Aß are still facing challenges on how to avoid interference of certain biological molecules and prevent triggering the activation of immune responses and blood clotting. Here, a biomimetic nanozyme (CuxO@EM-K) with augmented protein adsorption resistance, minimized immunogenicity, and enhanced biocompatibility is designed and synthesized. The CuxO@EM-K is made of CuxO nanozyme wrapped with modified 3xTg-AD mouse erythrocyte membrane with Aß-targeting pentapeptide KLVFF. KLVFF serves as Aß-specific ligand that works together with erythrocyte membrane to selectively capture Aß in the blood. Meanwhile, the erythrocyte membrane coating prevents protein coronas formation and thus retains Aß-targeting ability of CuxO@EM-K in biological fluids. More importantly, the CuxO core with multiple antioxidant enzyme-like activities stabilizes the outer erythrocyte membrane and simultaneously mitigates Aß-induced membrane oxidative damage, which enables the extended systemic circulation indispensable for adsorbing Aß. In vivo studies demonstrate that CuxO@EM-K not only reduces Aß burden in the blood and brain but also ameliorates memory deficits in the widely used 3xTg-AD mouse model. Moreover, CuxO@EM-K shows no apparent toxicity in 3xTg-AD mice. Overall, this work provides an example for developing biocompatible and synergistic clearance of peripheral Aß associated with AD.

Exploring and characterizing a novel combination of paeoniflorin and talatizidine for the treatment of rheumatoid arthritis.

Wutou Decoction (WTD) achieves favorable therapeutic response in treating rheumatoid arthritis (RA), especially for wind-cold-dampness stimulating RA. However, its material basis and molecular mechanisms remain unclear. To address this problem, the main bioactive compounds (BACs) of WTD against RA and the candidate targets were identified in the current study via transcriptional regulatory network analysis, computational structure-based methods, as well as in vivo and in vitro experimental validations. As a result, we successfully established a RA rat model named AIA-S, which simulated the clinical manifestations and pathological changes of wind-cold-dampness stimulating RA, and also displayed the distinctive characteristics and biological basis of inflammatory-immune system imbalance and abnormal energy metabolism changes. In addition, ALOX15B-PPAR-γ-PTGS2-FGF2-IL-1ß-c-JUN-MMP13-TGF-ß1 signal axis, involved into thermogenesis and energy metabolism, as well as maintaining the balance of inflammation-immune system, was identified as a candidate target of WTD against RA, according to the transcriptional regulatory network analysis on "RA-related gene-WTD-effective gene interaction network". Moreover, Paeoniflorin (PAE) and Talatizidine (TLT) were demonstrated to be the main BACs of WTD against RA for the following reasons: firstly, both PAE and TLT were the BACs of WTD according to ADME analysis in silico and the pharmacokinetics analysis in vivo. Secondly, both PAE and TLT were able to bind with PPAR-γ, c-JUN, MMP13 and TGF-ß1, which were the candidate targets of WTD against RA, with the strong binding affinity. Thirdly, the PAE and TLT combination exerted significant therapeutic effects on AIA-S rats through reversing the imbalance of inflammatory-immune system, and the disturbance of thermogenesis and energy metabolism, which were similar to WTD. More importantly, the administration of TLT or PAE alone didn't exert as prominently therapeutic effects as that of the two-BAC-combination did. Fourthly, the PAE and TLT combination promoted adipogenesis and lipogenesis by upregulating the PPAR-γ-induced lipogenic proteins. In conclusion, this study identified PAE and TLT as the main BACs of WTD in alleviating the severity of RA, and also developed a novel combination of PAE and TLT as a promising candidate drug for RA therapy.

Fecal metabolomics based on mass spectrometry to investigate the mechanism of qishen granules against isoproterenol-induced chronic heart failure in rats.

Qishen granules, derived from clinical experience formula, has been widely used to improve and treat myocardial ischemic chronic heart failure in China. However, the mechanism of action of Qishen granules in the treatment of chronic heart failure is unclear. This study aimed to discover potential biomarkers of isoproterenol-induced chronic heart failure rats and investigate the potential mechanism of Qishen granules treatment of chronic heart failure. The fecal metabolomics method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to analyze the therapeutic effect and metabolic changes of Qishen granules on chronic heart failure rats. Totally, 17 potential biomarkers were identified, involving bile acid metabolism, fatty acid metabolism, inflammatory response, and amino acid metabolism. For bile acid metabolism, we selected 12 bile acids (two of which were potential biomarkers in nontargeted metabolomics) for quantitative analysis. The quantitative results of bile acids showed that after Qishen granules treatment, the contents of bile acids such as ursodeoxycholic acid and glycodeoxycholic acid were similar to those of health group. This study helps to understand the pathogenesis of isoproterenol-induced chronic heart failure and the therapeutic mechanism of Qishen granules from the perspective of metabolic pathways.

A wide-targeted urinary and serum metabolomics strategy reveals the effective substance of the Wu-tou decoction.

As an important Chinese medicine decoction, Wu-tou decoction has been used to treat rheumatic arthritis for more than a thousand years. We previously reported that the Wu-tou decoction could change the urinary and serum metabolites in adjuvant-induced arthritis rats significantly. The purpose of this research was to confirm the potential biomarkers obtained by previous non-targeted metabolomics study through quantitative analysis by liqui chromatography with tandem mass spectrometry, in the meantime, to further study the effective material basis of Wu-tou decoction. Firstly, the important compounds in the tryptophan metabolism pathway, the arginine and proline metabolism pathway, the amino acid metabolism pathway, the tricarboxylic acid cycle, the vitamin B6 metabolism pathway, and the phenylalanine metabolism pathway, which were identified as potential biomarkers in previous study, were selected for quantitative analysis. Then the linearity, limit of detection, limit of quantification, selectivity, accuracy, precision, stability, recovery, and matrix effect of the quantitative method were examined. Finally, ten and eighteen metabolites were quantitatively analyzed in the serum and urine, respectively. The results showed that seven out of ten serum potential biomarkers and ten out of eighteen urine potential biomarkers were confirmed as real biomarkers. This research provides a powerful reference for the study on effective material basis of Wu-tou decoction.

A metabolomic study of the urine of rats with Alzheimer's disease and the efficacy of Ding-Zhi-Xiao-Wan on the afflicted rats.

As a well-known traditional Chinese medicine formula, Ding-Zhi-Xiao-Wan has long been used for the routine treatment of Alzheimer's disease. However, the mechanism of Ding-Zhi-Xiao-Wan in treating Alzheimer's disease is unclear. Therefore, a nontargeted metabolomics method based on ultrahigh performance liquid chromatography with quadrupole time-of-flight mass spectrometry has been established to explore the metabolic variations in the urine of Alzheimer's disease rats and investigate the therapeutic mechanism of Ding-Zhi-Xiao-Wan on Alzheimer's disease. To develop a better rat model of Alzheimer's disease, amyloid ß25-35 was injected into the bilateral hippocampus of Sprague-Dawley rats. Multivariate analysis approaches were applied to differentiate the urine components between the four groups. Thereafter, a targeted metabolomics method was used to verify the identified endogenous metabolites and determine the mechanism of action of Ding-Zhi-Xiao-Wan. Altogether, 26 potential biomarkers were found, of which 15 biomarkers (10 of which are potential biomarkers found in nontargeted metabolomics) were identified. The results show that Ding-Zhi-Xiao-Wan mainly affects the pathways of taurine and hypotaurine metabolism, tryptophan metabolism, and phenylalanine metabolism. Ding-Zhi-Xiao-Wan might play a role in the treatment of Alzheimer's disease by mediating antioxidative stress, regulation of energy metabolism, improvement of intestinal microbes, and protection of nerve cells.

pH-Zone-refining counter-current chromatography for two new lipo-alkaloids separated from refined alkaline extraction of Kusnezoff monkshood root.

An efficient and refined method for the separation of six aconitine-type alkaloids from the alkaline prepared "Kusnezoff monkshood root" was established. It is the first study that two new lipo-alkaloids were successfully isolated from refined sample by pH-zone-refining counter-current chromatography rather than synthetic method. It was of interest that a great deal of lipo-alkaloids was produced in crude extract from the alkalization of "Kusnezoff monkshood root." A refined sample method was proposed to enrich two types of alkaloids by liquid-liquid extraction, i.e. lipo-alkaloids and monoester-diterpenoid alkaloids. The pH-zone-refining counter-current chromatography was performed with an optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:5:4:5, v/v), where upper organic phase was added to 3 mmol/L triethylamine as a retainer and lower aqueous mobile phase was added to 3 mmol/L hydrochloric acid as an eluter. As a result, six aconitum alkaloids, including two lipo-alkaloids (8-lino-14-benzoylaconine, 8-pal-14-benzoylaconine), three monoester-diterpenoid alkaloids (14-benzoylmesaconine, 14-benzoylaconine, beyzoyldeoxyaconine), and one aconine alkaloid (neoline) were acquired from the plant at the same time. The anti-inflammatory activities of the two new lipo-alkaloids were compared to the six alkaloids in vitro, in cyclo-oxygen-ase-2 inhibition assays. The separation mechanism of six alkaloids by pH-zone-refining counter-current chromatography was illustrated.

Systematically Characterize the Anti-Alzheimer's Disease Mechanism of Lignans from S. chinensis based on In-Vivo Ingredient Analysis and Target-Network Pharmacology Strategy by UHPLC⁻Q-TOF-MS.

Lignans from Schisandra chinensis (Turcz.) Baill can ameliorate cognitive impairment in animals with Alzheimer's disease (AD). However, the metabolism of absorbed ingredients and the potential targets of the lignans from S. chinensis in animals with AD have not been systematically investigated. Therefore, for the first time, we performed an in-vivo ingredient analysis and implemented a target-network pharmacology strategy to assess the effects of lignans from S. chinensis in rats with AD. Ten absorbed prototype constituents and 39 metabolites were identified or tentatively characterized in the plasma of dosed rats with AD using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Based on the results of analysis of the effective constituents in vivo, the potential therapeutic mechanism of the effective constituents in the rats with AD was investigated using a target-network pharmacology approach and independent experimental validation. The results showed that the treatment effects of lignans from S. chinensis on cognitive impairment might involve the regulation of amyloid precursor protein metabolism, neurofibrillary tangles, neurotransmitter metabolism, inflammatory response, and antioxidant system. Overall, we identified the effective components of lignans in S. chinensis that can improve the cognitive impairment induced by AD and proposed potential therapeutic metabolic pathways. The results might serve as the basis for a fundamental strategy to explore effective therapeutic drugs to treat AD.

Equivalently Quantitative Ion Strategy with Quaternary Ammonium Cation Derivatization for Highly Sensitive Quantification of Lanostane-Type Triterpene Acids without Standards by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS).

Lanostane-type triterpene acids are one of the main active components in Poria cocos, which have strong anticancer, immune regulation, antiaging, and anti-inflammation effects. However, low abundance, structure similarity, poor ionization efficiency, and especially the lack of adequate standards has hampered their accurate quantification. In this work, we develop an equivalently quantitative ion strategy with quaternary ammonium cation derivatization and established only one calibration curve for highly sensitive quantification of lanostane-type triterpene acids by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in the absence of standards. Quaternary ammonium derivatives of all the lanostane-type triterpene acids generate two specific fragment ions of m/ z 130 and 170. Moreover, the signal intensity of the m/ z 170 fragment is positively correlated with the concentration of all derivatives under the optimal MS parameters, so this fragment is chosen as the equivalently quantitative ion to easily quantify derivatived lanostane-type triterpene acids. The concentration of derivatives and the signal intensity of m/ z 170 have a good linear relationship, with a correlation coefficient ( r2) of >0.999, and the good accuracy, precision, and high recovery guaranteed the reliability of lanostane-type triterpene acid derivative quantification. This derivatization method enables accurate quantitation of a total of 53 and 19 lanostane-type triterpene acids in the Poria cocos extracts and MDCK cell bidirectional transport samples with a limit of quantitation (LOQ) of 0.1 nM, respectively. This method also could be applied to quantitatively analyze the derivatives of the carboxyl compounds that have the same core structure with small differences in substituents.