Multifunctional thiolated chitosan/puerarin composite hydrogels with pH/glutathione dual responsiveness for potential drug carriers.

Puerarin (PUE), a natural and biologically active isoflavone extracted from Chinese medicine Pueraria lobata, can self-assemble to form a hydrogel without other chemical modifications. However, although PUE hydrogel has pH responsivity, but it is difficult to adapt to the changeable pathological environment. Therefore, thiolated chitosan (TCS) is synthesized and hybridized with PUE hydrogel to prepare TCS10/PUE composite hydrogel. The results of rheological measurement showed that the resultant composite hydrogels inherited the low loss performance of TCS hydrogel, which means that they have stronger elasticity. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images displayed that TCS10/PUE composite hydrogel has a fibrous-network structure. X-Ray Diffractometer (XRD) and Fourier transform infrared spectroscopy (FT-IR) proved the existence of hydrogen bonds and disulfide bonds in the formation of composite hydrogel. Degradation experiment showed that TCS10/PUE composite hydrogels have pH and glutathione (pH/GSH) dual sensitivity. Furthermore, TCS10/PUE composite hydrogels exhibited multi-functionality including thixotropy, cytocompatibility, antibacterial and anti-inflammatory properties. Berberine chloride hydrate (BCH) was further used as a model drug for in vitro release study. BCH and PUE could be released cooperatively under pH/GSH dual responsivity. These results indicated that the resultant composite hydrogel has eminent pH/GSH dual responsivity and could act as a potential new intelligent drug carrier.

Elucidating the mechanisms underlying the anti-hyperlipidemic effects of Laportea bulbifera using integrated serum metabolomics and network pharmacology.

Hyperlipidemia is a chronic metabolic disorder characterized by alterations in lipid metabolism as well as other pathways. Laportea bulbifera, an indigenous medicinal plant of Chinese herbal medicine, exhibits therapeutic effects on hyperlipidemia, but the mechanisms remain unclear. This study investigated the potential mechanisms underlying the anti-hyperlipidemic effects of L. bulbifera using an integrated strategy based on metabolomics and network pharmacology methods that were established to investigate the potential mechanism of anti-hyperlipidemia effect of L. bulbifera. First, the therapeutic effects of L. bulbifera on body weight reduction and biochemical indices were assessed. Next, 18 significant metabolites distinguishing the control and model groups were identified based on serum metabolomics and multivariate analyses. Then, a compound-target network was constructed by linking L. bulbifera and hyperlipidemia using network pharmacology. Three metabolic pathways involved in treating hyperlipidemia were identified. Finally, five crucial targets were selected by constructing a bionetwork starting from the compounds and ending in the metabolites. This study established an integrated strategy based on metabolomics coupled with network pharmacology and revealed the mechanism underlying the protective effects of L. bulbifera against hyperlipidemia for the first time.

Diet Restriction Impact on High-Fat-Diet-Induced Obesity by Regulating Mitochondrial Cardiolipin Biosynthesis and Remodeling.

Diet restriction (DR) ameliorates obesity by regulating mitochondrial function. Cardiolipin (CL), a mitochondrial phospholipid, is closely associated with mitochondrial function. This study aimed to evaluate the anti-obesity effects of graded levels of DR based on mitochondrial CL levels in the liver. Obese mice were treated with 0%, 20%, 40%, and 60% reductions in the normal diet compared to normal animals (0 DR, 20 DR, 40 DR, and 60 DR groups, respectively). Biochemical and histopathological analyses were performed to evaluate the ameliorative effects of DR on obese mice. The altered profile of mitochondrial CL in the liver was explored using a targeted metabolomics strategy by ultra-high-pressure liquid chromatography MS/MS coupled with quadrupole time-of-flight mass spectrometry. Finally, gene expression associated with CL biosynthesis and remodeling was quantified. Tissue histopathology and biochemical index evaluations revealed significant improvements in the liver after DR, except for the 60 DR group. The variation in mitochondrial CL distribution and DR levels showed an inverted U-shape, and the CL content in the 40 DR group was the most upregulated. This result is consistent with the results of the target metabolomic analysis, which showed that 40 DR presented more variation. Furthermore, DR led to increased gene expression associated with CL biosynthesis and remodeling. This study provides new insights into the mitochondrial mechanisms underlying DR intervention in obesity.

Dynamical changes of tea metabolites fermented by Aspergillus cristatus, Aspergillus neoniger and mixed fungi: A temporal clustering strategy for untargeted metabolomics.

Dark tea fermentation involves various fungi, but studies focusing on the mixed fermentation in tea remain limited. This study investigated the influences of single and mixed fermentation on the dynamical alterations of tea metabolites. The differential metabolites between unfermented and fermented teas were determined using untargeted metabolomics. Dynamical changes in metabolites were explored by temporal clustering analysis. Results indicated that Aspergillus cristatus (AC) at 15 days, Aspergillus neoniger (AN) at 15 days, and mixed fungi (MF) at 15 days had respectively 68, 128 and 135 differential metabolites, compared with unfermentation (UF) at 15 days. Most of metabolites in the AN or MF group showed a down-regulated trend in cluster 1 and 2, whereas most of metabolites in the AC group showed an up-regulated trend in cluster 3 to 6. The three key metabolic pathways mainly composed of flavonoids and lipids included flavone and flavonol biosynthesis, glycerophospholipid metabolism and flavonoid biosynthesis. Based on the dynamical changes and metabolic pathways of the differential metabolites, AN showed a predominant status in MF compared with AC. Together, this study will advance the understanding of dynamic changes in tea fermentation and provide valuable insights into the processing and quality control of dark tea.

Exploration of the hypoglycemic mechanism of Fuzhuan brick tea based on integrating global metabolomics and network pharmacology analysis.

Introduction: Fuzhuan brick tea (FBT) is a worldwide popular beverage which has the appreciable potential in regulating glycometabolism. However, the reports on the hypoglycemic mechanism of FBT remain limited. Methods: In this study, the hypoglycemic effect of FBT was evaluated in a pharmacological experiment based on Kunming mice. Global metabolomics and network pharmacology were combined to discover the potential target metabolites and genes. In addition, the real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed for verification. Results: Seven potential target metabolites and six potential target genes were screened using the integrated approach. After RT-qPCR analysis, it was found that the mRNA expression of VEGFA, KDR, MAPK14, and PPARA showed significant differences between normal and diabetes mellitus mice, with a retracement after FBT treatment. Conclusion: These results indicated that the hypoglycemic effect of FBT was associated with its anti-inflammatory activities and regulation of lipid metabolism disorders. The exploration of the hypoglycemic mechanism of FBT would be meaningful for its further application and development.

Sex Differences of Cardiolipin in Tissue Distribution Based on Targeted Lipidomic Analysis by UHPLC-QTOF-MS/MS.

Cardiolipins (CLs) are involved in ATP production, mitochondria biogenesis, apoptosis and mitophagy. Their tissue distribution can provide insight into the function of mitochondria and related diseases. However, the reports on tissue distribution of CLs remain limited. In this research, CLs were identified from heart, liver, kidney, spleen, lung, skeletal muscle, and brain using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS). Then, the distribution and sex difference of CLs in seven tissues were compared by a targeted lipidomic approach. A total of 88 CLs were identified, of which 58, 51, 57, 58, 50, 61 and 52 CLs were found in heart, liver, kidney, spleen, lung, skeletal muscle, and brain, respectively. Compared with the distribution of CLs in heart, liver, kidney, and skeletal muscle, the CLs in spleen, lung, and brain showed significant differences. Moreover, the results indicated that there were sex differences of CLs in liver and kidney. A total of 16 CLs in liver tissue and 21 CLs in kidney tissue, with significant sex differences, were screened. Our findings in the targeted lipidomic analysis demonstrated that tissue distribution of CLs was essential in the dynamic states and sex differences of CLs, which might provide evidence for the mitochondrial-related mechanism under physiological and pathological conditions.

An injectable supramolecular nanofiber-reinforced chitosan hydrogel with antibacterial and anti-inflammatory properties as potential carriers for drug delivery.

The inherent weak mechanical strength of chitosan physical cross-linking hydrogels (CS hydrogels) limits their applications in biomaterials. Hence, puerarin (PUE) as a self-assembly active small molecule in herbal was introduced in CS hydrogels to fabricate CS/PUE18 composite hydrogels with interpenetrating network structure. The result of rheological measurement showed that storage modulus and loss modulus of CS/PUE18 composite hydrogels were improved by three orders of magnitude, indicating that the introduction of PUE significantly reinforced CS hydrogels. The results of SEM and BET measurement demonstrated that macromolecular chains of CS intertwined with nanofibers of PUE, which caused the network structure of CS/PUE18 composite hydrogels to become denser. XRD patterns and FT-IR spectra verified that the amino groups in CS formed hydrogen bonding with the hydroxyl groups in PUE. Degradation and swelling experiments showed that CS/PUE18 composite hydrogels have pH sensitivity. Moreover, CS/PUE18 composite hydrogels exhibited multi-functionality including injectability, thixotropy, cytocompatibility, antibacterial and anti-inflammatory properties. The release behavior of berberine chloride hydrate (BCH) and PUE from the resultant CS/PUE18 composite hydrogels have pH dependence. These results revealed that injectable CS/PUE18 composite hydrogels with dual antibacterial and anti-inflammatory properties could be potential delivery vehicles for sustained and controlled release.

Potential hypoglycemic metabolites in dark tea fermented by Eurotium cristatum based on UPLC-QTOF-MS/MS combining global metabolomic and spectrum-effect relationship analyses.

The preventive and therapeutic effects of dark tea fermented by Eurotium cristatum (DTE) in glucose metabolism have been demonstrated. However, few studies have investigated comprehensive changes in the chemical composition and activity in DTE before and after fermentation. In this study, the metabolic profiling of raw samples and fermented samples was determined by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). Furthermore, a systematic analytical strategy combining global metabolomics and the spectrum-effect relationship based on α-glucosidase inhibition was employed for screening discriminant metabolites. As a result, 15 discriminant metabolites were identified in DTE samples. Among them, 10 metabolites (4 fatty acids, 1 dyphylline derivative, 3 lysophosphatidylcholines, and 2 triterpenes) increased in relative contents and the contents of the other 5 polyphenol metabolites decreased after fermentation. These metabolites were critical constituents possibly associated with DTE's hypoglycemic activity, which also might be suitable as quality evaluation indicators. This study provided a worthy insight into the exploration of representative active constituents or quality indicators of DTE.

An integrated strategy for the establishment of a protoberberine alkaloid profile: Exploration of the differences in composition between Tinosporae radix and Fibraurea caulis.

INTRODUCTION: Accurate species and content identification of major active components in herbals are the guarantee of the safety and effectiveness for medical and commodity purposes. OBJECTIVES: In this study, an integrated strategy used to establish the protoberberine alkaloid profile was applied to explore the differences in composition between the pieces of Tinosporae radix and Fibraurea caulis, both of which had morphological similarities. MATERIALS AND METHODS: First, an in-house library including possible protoberberine alkaloids based on different substituents was predicted by systematic literature survey. Meanwhile, diagnostic fragments of protoberberine alkaloids were investigated using the corresponding standards. Second, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was used to obtain multidimensional mass spectral data. Then, the identifications were confirmed by targeted filter of the acquired data based on the library. RESULTS: As a result, 10 protoberberine alkaloid molecules including 46 isomers were identified or characterised. The qualitative distribution and relative content of protoberberine alkaloids revealed the fundamental difference between Tinosporae radix and Fibraurea caulis. 25 alkaloids were present in both herbals, while five compounds were detected only in Tinosporae radix. Furthermore, the contents of four alkaloids in Tinosporae radix were significantly higher than those in its adulterant, Fibraurea caulis. CONCLUSION: The five unique ingredients in Tinosporae radix can be used as a better indicator for distinguishing the pieces of Tinosporae radix and Fibraurea caulis. The protoberberine alkaloid profile established in this study can be applied to quality evaluation of the two herbals or other herbals containing major protoberberine alkaloids.

Changes in Triterpenes in Alismatis rhizoma after Processing Based on Targeted Metabolomics Using UHPLC-QTOF-MS/MS.

Alismatis rhizoma (AR) has been used as an herbal medicine in China for over a thousand years. Crude AR, salt-processed AR (SAR), and bran-processed AR (BAR) are recorded in the Pharmacopoeia of the People's Republic of China. However, the differences of chemical composition between crude AR and its processing products remain limited. In this study, triterpenes were identified from crude AR, SAR, and BAR by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometer (UHPLC-QTOF-MS/MS). Subsequently, the differences of triterpenes between the crude AR and processed ARs were compared via a targeted metabolomics approach. Finally, a total of 114 triterpenes were identified, of which 83, 100, and 103 triterpenes were found in crude AR, SAR, and BAR, respectively. After salt-processing, there were 17 triterpenes newly generated, 7 triterpenes with trends of increasing, and 37 triterpenes decreased. Meanwhile, 56 triterpenes including 21 newly generated and 35 with significant increases were observed in BAR. This study could be benefit to investigate the processing mechanism of AR, as well as support their clinical applications.

N-(9-Fluorenylmethoxycarbonyl)-L-Phenylalanine/nano-hydroxyapatite hybrid supramolecular hydrogels as drug delivery vehicles with antibacterial property and cytocompatibility.

The intrinsic fragility of hydroxyapatite (HAP) restricts its wider applications for local delivery of antibiotics. The composites formed by integrating HAP with hydrogels can improve the properties of HAP. However, these reported composites not only require tedious preparation and employ organic solvent and toxic reagents, but also hardly have inherent antimicrobial property. In this study, N-(9-Fluorenylmethoxycarbonyl)-L-Phenylalanine/nano-hydroxyapatite (Fmoc-L-Phe/nHAP) hybrid supramolecular hydrogels with antibacterial property and cytocompatibility was prepared by integrating nHAP as reinforcement with Fmoc-L-Phe supramolecular hydrogels. The results showed that nHAP bounds in the chamber of the gel network and adheres to the fiber of Fmoc-L-Phe due to intermolecular interaction, remarkably improving the mechanical strength of Fmoc-L-Phe supramolecular hydrogels. The results of inhibition zone experiment and MTT experiment showed that the Fmoc-L-Phe/nHAP hybrid supramolecular hydrogels possess antimicrobial property and cytocompatibility. In vitro release experiment of chlorogenic acid (CGA) from the hybrid supramolecular hydrogels was performed. The study of the release kinetics indicated that the release behavior of CGA from the hybrid supramolecular hydrogels is following Weibull model and release mechanism involved Fickian diffusion and erosion of the surface of hydrogel matrix. The release of CGA shows a good inhibition effect on S. aureus. The results show that the Fmoc-L-Phe/nHAP hybrid hydrogels with antibacterial property and cytocompatibility have promising applications as drug delivery carrier. Due to the intrinsic fragility of hydroxyapatite (HAP), the properties of HAP could be improved by incorporation into hydrogels. However, these reported composites not only require tedious preparation and employ organic solvent and toxic reagents, but also hardly have inherent antimicrobial property. We prepared N-(9-Fluorenylmethoxycarbonyl)-L-Phenylalanine/nano-hydroxyapatite (Fmoc-L-Phe/nHAP) hybrid supramolecular hydrogels by integrating nHAP as reinforcement with Fmoc-L-Phe supramolecular hydrogels. The results showed that nHAP bounds in the chamber of the gel network and adheres to the fiber of Fmoc-L-Phe due to intermolecular interaction, remarkably improving the mechanical strength of Fmoc-L-Phe supramolecular hydrogels. The results of inhibition zone experiment and MTT experiment showed that the Fmoc-L-Phe/nHAP hybrid supramolecular hydrogels possess antibacterial property and cytocompatibility. In vitro release experiment of chlorogenic acid (CGA) from the hybrid supramolecular hydrogels was performed. The study of the release kinetics indicated that the release behavior of CGA from the hybrid supramolecular hydrogels is following Weibull model and release mechanism involved Fickian diffusion and erosion of the surface of hydrogel matrix. The release of CGA shows a good inhibition effect on S. aureus. The results show that the Fmoc-L-Phe/nHAP hybrid hydrogels with antibacterial property and cytocompatibility have promising applications as drug delivery carrier.

A novel predict-verify strategy for targeted metabolomics: Comparison of the curcuminoids between crude and fermented turmeric.

Curcuminoids are the major bioactive constituents of turmeric, the application of which are limited by the poor bioavailability. In this study, turmeric was fermented by the Monascus purpureus and Eurotium cristatum fungi to enhance its bioavailability. To explore the variations in the curcuminoids contents in fermented turmeric, a targeted predict-verify strategy was established. For targeted analysis of curcuminoids, a compound library containing all possible curcuminoids based on their structural skeleton was predicted and built for targeted scanning. Then, the MS data were automatically matched with the predicted library to verify the corresponding curcuminoids. As a result, 115 curcuminoids (48 novel compounds and 14 compounds reported in turmeric for the first time) were fully characterized in crude and fermented turmeric. Among these curcuminoids, 31 were newly generated in fermented turmeric. The established predict-verify strategy allows for an efficient and automatic metabolomic analysis to screen for curcuminoids with potentially better bioavailability.

The metabolic change in serum lysoglycerophospholipids intervened by triterpenoid saponins from Kuding tea on hyperlipidemic mice.

Triterpenoid saponins from Kuding tea have demonstrated preventive effects on hyperlipidaemia induced by a high-fat diet. Lysoglycerophospholipids (Lyso-GPLs) are known to be associated with proatherogenic conditions such as hyperlipidaemia. In this study, a target profiling strategy based on a multiple reaction monitoring mode was applied for the analysis of Lyso-GPLs. The metabolic changes were evaluated by the qualitative and relative quantitative distribution of six classes of Lyso-GPLs in mouse serum. A total of 153 Lyso-GPL regioisomers, consisting of 85 lysophosphatidylcholines, 15 lysophosphatidic acids, 23 lysophosphatidylethanolamines, 5 lysophosphatidylserines, 19 lysophosphatidylinositols and 6 lysophosphatidylglycerols, were detected and quantified. The results showed decreased trends in the content of total Lyso-GPLs in the serum of hyperlipidemic mice compared with that in normal controls. The content of total Lyso-GPLs significantly increased after treatment with triterpenoid saponins from Kuding tea. Among them, the proportions of most Lyso-GPLs with a higher degree of unsaturation or a longer carbon chain in fatty acyl chains dramatically decreased in hyperlipidemic mice. However, this tendency reversed after the treatment of triterpenoid saponins from Kuding tea. This is the first study regarding a target profiling strategy for the quantitative analysis of six different types of Lyso-GPLs on high-fat diet-induced hyperlipidemic mice intervened by Kuding tea. Those Lyso-GPLs changed significantly may be potential biomarkers for hyperlipidaemia, and involved in the mechanism of the preventive intervention of Kuding tea on Lipid metabolic diseases.

Anti-Asthma Effect of an Active Components Group from Decoction of Pheretima Aspergillum and Its Chemical Composition Characterized by Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry.

The decoction of Pheretima aspergillum is used as a traditional medicine for treatment of asthma in China with a long clinical history. The aim of the study was to investigate the anti-asthma activities of an active components group obtained from the decoction of Pheretima aspergillum in histamine and ovalubumin induced asthma guinea pigs. The experimental asthma model was established with spray of histamine and ovalbumin (OVA) followed by intragastric administration of Pheretima aspergillum extract and fractions. The incubation period of asthma, serum IFN-γ, IL-4, and LTB4 levels were tested and determined. In addition, liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) was used to identify the bioactive components in active fraction. The significant increase of asthma incubation period, serum IFN-γ level were observed in the asthma guinea pigs treated with the active components group. The serum IL-4 and LTB4 levels were obviously decreased compared to that of model controls. In addition, twenty oligopeptides were first identified or characterized in the active fraction from the decoction of Pheretima aspergillum. The results indicated that Pheretima aspergillum can inhibit infiltration and diffusion of inflammatory cells in asthma model guinea pigs, and regulate IFN-γ, IL-4, and LTB4 secretions. It is reasonable to assume that the anti-asthma activities of Pheretima aspergillum mainly resulted from these oligopeptides. Also, synergistic effects among their compounds may exist in the anti-asthma activity of Pheretima aspergillum.

Enrichment and Cytotoxic Activity of Curcuminoids from Turmeric Using Macroporous Resins.

Curcuminoids are functional secondary metabolites abundant in turmeric. In the present study, a simple and efficient method for enrichment of curcuminoids from turmeric was developed using macroporous resin. Eight different types of macroporous resins were examined by static adsorption/desorption properties and the type of XDA-7 was selected as the optimum one. Under the optimized conditions, the final contents of refined extract excluded 84.2% of impurities, by comparison with crude extract in a scale-up experiment. Meanwhile, 8 representative curcuminoids including 4 dihydro- and tetrahydro-curcuminoids were enriched, isolated, and identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) data. In addition, the individual curcuminoids were prepared to evaluate their cytotoxic activity toward HeLa tumor cell lines. All compounds, especially the trace amount of curcuminoids, demonstrated notable cytotoxic activity. The results supported that those trace amount of curcuminoids can be good candidates for drug development as anticancer agents. The purification process was simple and efficient, which could afford a potential method to enrich and concentrate not only the major curcuminoids, but also the trace amount of ones from turmeric raw materials for industrial applications.

An integrated strategy for establishment of metabolite profile of endogenous lysoglycerophospholipids by two LC-MS/MS platforms.

The methods for detecting endogenous phospholipids (PL) are focused on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since the structural relation and similar core structure of glycerophospholipids results in a common and predictable mass spectrometric fragmentation behavior, in the present study, six classes of lysoglycerophospholipid (Lyso-PL) were subjected to a predict-verify approach by multiple reaction monitoring (MRM) mode based on liquid chromatography-quadrupole linear ion trap mass spectrometry (LC-QTRAP-MS/MS) complemented with liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) in biological serum samples. A targeted metabolite profile of Lyso-PL was established and represented in the form of a LC-QTRAP-MRM analysis. There were total of 96 Lyso-PL molecular species, consisting of 317 regioisomers detected in serum of human and four rodent species. In addition, the metabolite profile was successfully applied to the analysis of qualitative distribution of Lyso-PLs in serum of human and four rodent species. We believe that this improvement in the method for qualitative analysis of endogenous Lyso-PLs should greatly contribute to identifying the role of lipid molecular species in biological systems. Also, the integrated strategy for establishment of metabolite profile can be applied to targeted qualitative analysis of other endogenous metabolites which occur in a high individual number but sharing either structural similarities or similar functional groups.

[Identification of major components of Xingsusan decoction by HPLC-HR-TOF/MS].

OBJECTIVE: The major components of Xingsusan decoction were studied by HPLC-HR-TOF-MS. METHODS: A Welchrom- C18 column (4.6 mm x 250 mm, 5 µm) was used at a flow rate of 1.0 mL/min. The HPLC gradient was acetonitrile-formic acid (99.9: 0.1, V/V) (B) and water-formic acid (99.9:0.1, V/V) (A). HR-MS/MS analysis were carried out using a Micro/TOF-Q II Focus mass spectrometer fitted with an ESI source operating in Auto-MSn mode to obtain fragment ions. TIC chemical profiles of the extract were established in both positive and negative modes. The structures of the known components in Xingsusan decoction were identified by comparing their retention times, molecular weight and CID fragmentation patterns with their corresponding compounds reported in the literature. RESULTS: A total of 52 components were simultaneously detected in Q-TOF/MS. CONCLUSION: This method can be used as reference for other Chinese Medicine formulas study.