Mulberry (Morus alba L.) leaf water extract attenuates type 2 diabetes mellitus by regulating gut microbiota dysbiosis, lipopolysaccharide elevation and endocannabinoid system disorder.

ETHNOPHARMACOLOGICAL RELEVANCE: Mulberry (Morus alba L.) leaf is a well-known herbal medicine and has been used to treat diabetes in China for thousands of years. Our previous studies have proven mulberry leaf water extract (MLWE) could improve type 2 diabetes mellitus (T2D). However, it is still unclear whether MLWE could mitigate T2D by regulating gut microbiota dysbiosis and thereof improve intestinal permeability and metabolic dysfunction through modulation of lipopolysaccharide (LPS) and endocannabinoid system (eCBs). AIM OF STUDY: This study aims to explore the potential mechanism of MLWE on the regulation of metabolic function disorder of T2D mice from the aspects of gut microbiota, LPS and eCBs. MATERIALS AND METHODS: Gut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. LPS, N-arachidonoylethanolamine (AEA) and 2-ararchidonylglycerol (2-AG) contents in blood were determined by kits or liquid phase chromatography coupled with triple quadrupole tandem mass spectrometry, respectively. The receptors, enzymes or tight junction protein related to eCBs or gut barrier were detected by RT-PCR or Western blot, respectively. RESULTS: MLWE reduced the serum levels of AEA, 2-AG and LPS, decreased the expressions of N-acylphophatidylethanolamine phospholipase D, diacylglycerol lipase-α and cyclooxygenase 2, and increased the expressions of fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), alpha/beta hydrolases domain 6/12 in the liver and ileum and occludin, monoacylglycerol lipase and cannabinoid receptor 1 in the ileum of T2D mice. Furthermore, MLWE could change the abundances of the genera including Acetatifactor, Anaerovorax, Bilophila, Colidextribacter, Dubosiella, Gastranaerophilales, Lachnospiraceae_NK4A136_group, Oscillibacter and Rikenella related to LPS, AEA and/or 2-AG. Moreover, obvious improvement of MLWE treatment on serum AEA level, ileum occludin expression, and liver FAAH and NAAA expression could be observed in germ-free-mimic T2D mice. CONCLUSION: MLWE could ameliorate intestinal permeability, inflammation, and glucose and lipid metabolism imbalance of T2D by regulating gut microbiota, LPS and eCBs.

Mulberry leaf water extract alleviates type 2 diabetes in mice via modulating gut microbiota-host co-metabolism of branched-chain amino acid.

Elevations in circling branched-chain amino acids (BCAAs) levels associated with insulin resistance and type 2 diabetes mellitus (T2DM). Morus alba L. water extracts (MLE) show hypoglycemic function, but the precise mechanism remains obscure. This study is designed to investigate the association of the antidiabetes effect of MLE with the BCAAs co-metabolism modulated by host and gut microbiota. Tissue-specific expressions of BCAA-catabolizing enzymes were detected by RT-PCR and western blot, respectively. The components of the intestinal microflora were analyzed by high-throughput 16S rRNA gene sequencing. The results showed that MLE administration improved blood glucose and insulin level, decreased inflammatory cytokines expression, and lowered serum and feces BCAAs levels. Furthermore, MLE reversed the abundance changes of the bacterial genera correlated with serum and feces BCAAs, such as Anaerovorax, Bilophila, Blautia, Colidextribacter, Dubosiella, Intestinimonas, Lachnoclostridium, Lachnospiraceae_NK4A136, Oscillibacter, and Roseburia. Functionality prediction indicated that MLE potentially inhibited bacterial BCAAs biosynthesis, and promoted the tissue-specific expression of BCAAs catabolic enzyme. More importantly, MLE had obvious impacts on BCAA catabolism in germ-free-mimic T2DM mice. Those results indicated that MLE improving T2DM-related biochemical abnormalities is associated with not only gut microbiota modification but also the tissue-specific expression of BCAAs catabolic enzyme.

Amelioration of lipid accumulations and metabolism disorders in differentiation and development of 3T3-L1 adipocytes through mulberry leaf water extract.

BACKGROUND: Obesity is a worldwide problem that resulted from the excessive fat accumulation in adipose tissue, leading to the impairment of individual health. Mulberry leaf is an important traditional Chinese medicine and has been used to alleviate obesity for a long term. However, its underlying molecular mechanisms have not been fully elucidated yet. PURPOSE: In this study, we aimed to investigate the inhibition effects of mulberry leaf water extract (MLWE) on lipid accumulation during the process of differentiation of 3T3-L1 preadipocytes and development of mature adipocytes through the combination of molecular biology assays and metabolomic analysis. METHODS: The quality consistency and main chemical ingredients of MLWE were analyzed by high performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), respectively. Oil red O staining was used to mirror lipid accumulation. Lipogenesis-, lipolysis- and inflammation-related genes were evaluated by real-time PCR and western blot, respectively. Untargeted metabolomics were performed by LC-MS/MS. RESULTS: Prepared method and quality of MLWE were stable and reliable. A total of 34 compounds were identified and 14 of them were undoubtedly confirmed. MLWE supplementation could dose-dependently inhibit the aggregation of lipid droplets, and the expressions of sterol regulatory element-binding protein (SREBP)-1c, peroxisome proliferator-activated receptor (PPAR) γ, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), tumor necrosis factor (TNF)-α and interleukin (IL)-6, and increase the expressions of adenosine monophosphate-activated protein kinase (AMPK), hormone-sensitive lipase (HSL) and IL-10 in the differentiation of preadipocytes. Furthermore, MLWE treatment could dose-dependently decrease the level of triglycerides and the expressions of ACC, FAS, TNF-α, and IL-6, and up-regulate the level of glycerol and the expressions of PPARα, adiponectin (ADPN), adiponectin receptor (AdipoR) 1, AdipoR2, AMPK, HSL, and IL-10 in the development of mature adipocytes. Untargeted metabolomics showed that a total of 5 and 18 differential metabolites were reversed by MLWE intervention in the differentiation of preadipocytes and the development of mature adipocytes, respectively, which involved in the biosynthesis of unsaturated fatty acids, arachidonic acid metabolism and glycerophospholipids metabolism. CONCLUSION: Taken together, this study firstly verified that MLWE could effectively alleviate lipid accumulation and inflammation by regulating ADPN/AMPK-mediated signaling pathways and relevant metabolic disturbances including biosynthesis of unsaturated fatty acids, arachidonic acid metabolism and glycerophospholipids metabolism.

Morus alba L. water extract changes gut microbiota and fecal metabolome in mice induced by high-fat and high-sucrose diet plus low-dose streptozotocin.

Gut microbiota plays a key role in the pathophysiology of type 2 diabetes mellitus (T2D). Mulberry leaf has a hypoglycemic effect, but the potential mechanism is not fully understood. This study aimed to explore the influences and potential mechanisms of mulberry leaf water extract (MLWE) intervention on mice with T2D induced through a high-fat and high-sucrose diet combined with streptozotocin by the combination of fecal metabolomics and gut microbiota analysis. Results showed that MLWE could decrease fasting blood glucose and body weight while ameliorating lipid profiles, insulin resistance, liver inflammation, and the accumulation of lipid droplets in T2D mice. MLWE could reverse the abundances of the phyla Actinobacteria and Bacteroidetes and the ratio of Firmicutes/Bacteroidetes, and increase the abundances of the phyla Cyanobacteria and Epsilonbacteraeota in the feces of T2D mice. The abundances of genera Alloprevotella, Parabacteroides, Muribaculaceae, and Romboutsia in the feces of T2D mice could be reversed, while Oscillatoriales_cyanobacterium and Gastranaerophilales could be reinforced by MLWE supplementation. The levels of nine metabolites in the feces of T2D mice were improved, among which glycine, Phe-Pro, urocanic acid, phylloquinone, and lactate were correlated with Romboutsia and Gastranaerophilales. Taken together, we conclude that MLWE can effectively alleviate T2D by mediating the host-microbial metabolic axis.

Off-line two-dimensional liquid chromatography coupled with diode array detection and quadrupole-time of flight mass spectrometry for the biotransformation kinetics of Ginkgo biloba leaves extract by diabetic rat liver microsomes.

Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (Km) value of genistein was obvious increased while its maximal velocity (Vmax) and intrinsic clearance (CLint) values were significantly decreased by diabetic RLMs, and the Vmax and CLint values of kaempferol and isorhamnetin were notably enhanced while their Km values were markedly reduced. For the half-time (t1/2) values of four target compounds and the Km, Vmax and CLint values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE.

Mechanisms of rice straw biochar effects on phosphorus sorption characteristics of acid upland red soils.

An important pathway for biochar to alter the availability of soil phosphorus (P) is to change P sorption characteristics of the soil. The aim of this study was to understand the mechanisms of biochar effects on P sorption in acid upland red soils in the presence of different concentrations of exogenous P. Rice straw biochar (RSB) was prepared and applied at rates of 0, 1%, 3%, and 5% (w/w) to three red soils (MZ1, MZ2, and QY1) differing in initial pH (pH = 4.31, 4.82, and 5.68, respectively). The P sorption characteristics of these red soils were described using the Langmuir and Temkin equations and their relationships with soil basic physicochemical properties were analyzed. Furthermore, a representative red soil (MZ2) was selected to analyze the zeta potential of soil colloids and the chemical properties of sorption equilibrium solution, in order to understand their relationships with P sorption characteristics. Results showed that within a certain range of P concentration in the equilibrium solution, the amount of P sorbed by the three red soils decreased and the corresponding amount of P desorbed increased with increasing amendment rate of RSB. RSB showed the greatest effect on P desorption characteristics of MZ2 soil in the presence of higher exogenous P concentration. With increasing RSB amendment rate, the maximum P sorption of MZ1 soil decreased, while those of MZ2 and QY1 soils increased after an initial decrease. Phosphate sorption equilibrium constant and maximum P buffer capacity of each soil first increased and then decreased. However, a single physicochemical property could not interpret complex changes in multi-factors that jointly determine the P sorption characteristics of red soils. In the case of MZ2 soil, RSB amendment shifted the zeta potential of soil colloids to the negative direction; this decreased the positive charge and increased the negative charge on the soil surface, thus reducing P sorption in the MZ2 soil. In the presence of the same concentration of exogenous P, RSB amendment altered the pH, dissolved organic C (DOC), humification index (HIX), and maximum fluorescence intensity (Fmax) in the sorption equilibrium solution. In most cases, the amount of P sorbed by the MZ2 soil was negatively correlated with the pH value, DOC concentration, HIX value, and Fmax value of humic-like dissolved organic matter (DOM), and positively correlated with the Fmax value of protein-like DOM (P < 0.05 or P < 0.01). The relative fractional distribution of the contents for humic-like and protein-like DOM might determine the difference in the P sorption characteristics of MZ2 soil. In conclusion, different amendment rates of RSB affected the release of phosphate from soil surfaces into the solution by altering basic physicochemical and electrochemical properties of red soils and chemical properties of sorption equilibrium solution.

Simultaneous determination of baicalin, oroxylin A-7-O-glucuronide and wogonoside in rat plasma by UPLC-DAD and its application in pharmacokinetics of pure baicalin, Radix Scutellariae and Yinhuang granule.

A novel UPLC-DAD method was developed and validated for the simultaneous determination of baicalin (baicalein-7-glucuronide, BG), oroxylin A-7-O-glucuronide (OAG) and wogonoside (WG) in rat plasma using rutin as the internal standard. Plasma samples were precipitated using acetonitrile containing 0.1% formic acid. Separation was performed on an Agilent Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) using gradient acetonitrile and 0.2% formic acid water solution as mobile phase. The flow-rate was set at 0.4 mL/min and the eluate was detected at 275 nm. The method was linear over the ranges of 0.075-17.50, 0.050-12.60 and 0.056-14.10 µg/mL for BG, OAG and WG, respectively. The intra- and inter-day precisions were respectively <4.8% and 6.4%. All of the limits of detection of three analytes in rat plasma were 0.01 µg/mL, whereas the limits of quantification were, respectively, 0.035, 0.025 and, 0.025 µg/mL. This assay has been successfully applied to pharmacokinetics of BG, OAG and WG in rats after oral administration of Yinhuang granule (YHG) and comparative pharmacokinetics of BG in rats following oral administration of the pure BG, Radix Scutellariae (RS) or YHG. We speculate that some co-existing ingredients in RS or YHG may increase the absorption and elimination of BG in rat. This work may be helpful for the quality control of Yinhuang granule.

Screening for Potential Bioactive Components in Ginkgo biloba Extract by the Rat Renal Tubular Epithelial Cell Extraction and LC-MS/MS.

Rat renal tubular epithelial cell (RTEC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN). The compounds in GBE binding with cell membrane or entering into cell are still unknown, which may be potential bioactive components. In this paper, a powerful method for screening and analyzing the potential bioactive components from GBE was developed using cell extraction coupled with high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). 8 prototype compounds and 5 metabolites were obtained, among which 6 prototype compounds and 1 metabolite were identified or tentatively characterized as rutin, bilobalide, ginkgolide B, ginkgolide C, genkwanin, apigenin and diosmetin by comparing their retention times and MS spectra with those of authentic standards or literature data. The 6 prototype compounds were further quantitatively analyzed using electrospray ionization in negative mode multiple reaction monitoring (MRM). The results showed that high glucose changed the Tmax, MRT(0-t), Cmax and AUC(0-t) of all observed compounds and decreased the t1/2 of genkwanin and apigenin, significantly. The overall findings indicate that 8 prototype compounds may be the potential bioactive components of GBE with preventive effect against DN and the method of RTEC extraction coupled with LC-MS/MS technology screening method we developed is a feasible, rapid, and useful tool for screening and analyzing potential bioactive components.

Target cell extraction coupled with LC-MS/MS analysis for screening potential bioactive components in Ginkgo biloba extract with preventive effect against diabetic nephropathy.

A rapid and useful approach for screening potential bioactive components in Ginkgo biloba extract (GBE) with preventive effect against diabetic nephropathy (DN) was developed using mesangial cells extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Mesangial cells were first divided into two groups according to their treatments with high glucose or high glucose plus GBE. After incubation for 4, 8, 12, 16, 24 and 48 h, the cells were harvested and extracted with 40% acetic acid in water before LC-MS/MS analysis. Then, 19 compounds and five metabolites were found to selectively combine with mesangial cells. Notably, compounds including quercetin and rutin were identified or tentatively characterized according to the results of retention time and MS spectra, which is highly consistent with our previous reports that quercetin and rutin are potent protective agents against glomerulosclerosis in DN. Therefore, all these results indicate that target cell extraction coupled with LC-MS/MS analysis can be successfully applied for predicting the bioactive components in GBE with preventive effect against DN.

Two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to study the excretion and metabolic interaction of edaravone and taurine in rats.

In this study, two independent and complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the determination of edaravone or taurine in rat urine, feces and bile after intravenous administration, using 3-methyl-l-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Edaravone was separated on an Agilent Eclipse Plus C18 column (100×2.1 mm, 3.5 µm) using methanol and water (containing 5 mM ammonium formate and 0.02% formic acid) as mobile phase, while taurine was performed on a Waters Atlantis HILIC Silica column (150×2.1 mm, 3 µm) using acetonitrile and water (containing 5mM ammonium formate and 0.2% formic acid) as mobile phase. The mass analysis was performed in a Triple Quadrupole mass spectrometer via multiple reaction monitoring (MRM) with negative ionization mode. The optimized mass transition ion pairs (m/z) for quantification were 173.1→92.2 and 187.2→106.0 for edaravone and its IS, 124.1→80.0 and 172.0→80.0 for taurine and its IS, respectively. The validated methods have been successfully applied to the excretion and metabolism interaction study of edaravone and taurine in rats after independent intravenous administration and co-administration with a single dose. The results demonstrated that there were no significant alternations on the metabolism and cumulative excretion rate of edaravone and taurine, implying that the proposed combination therapy was pharmacologically viable.