Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Fabrication of a surface molecularly imprinted polymer membrane based on a single template and its application in the separation and extraction of phenytoin, phenobarbital and lamotrigine.

An innovative molecularly imprinted polymer membrane (MIPM) was prepared with polyvinylidene difluoride (PVDF) as the support, phenytoin (PHT) as the single template, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross-linking reagent, azobisisobutyronitrile as the initiator, and acetonitrile-dimethylformamide (1 : 1.5, v/v) as the porogen. These materials were characterized via scanning electron microscopy, Fourier transform infrared spectroscopy, Brunauer-Emmett-Teller measurements and X-ray photoelectron spectroscopy. Their adsorption performances were evaluated through a series of experiments including isothermal adsorption, kinetic adsorption, selective adsorption, adsorption-desorption, reusability, and preparation reproducibility. Additionally, the application was explored by investigating the extraction recovery of MIPMs towards PHT, phenobarbital (PHB) and lamotrigine (LTG) in different matrices including methanol, normal saline (NS), phosphate buffer solution (PBS) and plasma. The results showed that MIPMs with rough and porous surfaces were successfully constructed, which offered good preparation reproducibility, reusability and selectivity. The adsorption capacities of MIPMs towards PHT, PHB and LTG were 2.312, 2.485 and 2.303 mg g-1, respectively, while their corresponding imprinting factors were 8.538, 12.122 and 4.562, respectively. The adsorption equilibrium of MIPMs was achieved within 20 min at room temperature without stirring or ultrasonication. The extraction recoveries of MIPMs for PHT, PHB or LTG in methanol, NS and PBS were more than 80% with an RSD% value of less than 3.64. In the case of plasma, the extraction recovery of MIPMs for PHT and PHB was more than 80% with an RSD% value of less than 2.41, while that of MIPMs for LTG was more than 65% with an RSD% value of less than 0.99. All the results indicated that the preparation method for MIPMs was simple, stable, and reliable, and the prepared MIPMs possessed excellent properties to meet the extraction application of PHT, PHB and LTG in different matrices.

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.

Identification of geographic origins of Morus alba Linn. through surfaced enhanced Raman spectrometry and machine learning algorithms.

The leaves of Morus alba Linn., which is also known as white mulberry, have been commonly used in many of traditional systems of medicine for centuries. In traditional Chinese medicine (TCM), mulberry leaf is mainly used for anti-diabetic purpose due to its enrichment in bioactive compounds such as alkaloids, flavonoids and polysaccharides. However, these components are variable due to the different habitats of the mulberry plant. Therefore, geographic origin is an important feature because it is closely associated with bioactive ingredient composition that further influences medicinal qualities and effects. As a low-cost and non-invasive method, surface enhanced Raman spectrometry (SERS) is able to generate the overall fingerprints of chemical compounds in medicinal plants, which holds the potential for the rapid identification of their geographic origins. In this study, we collected mulberry leaves from five representative provinces in China, namely, Anhui, Guangdong, Hebei, Henan and Jiangsu. SERS spectrometry was applied to characterize the fingerprints of both ethanol and water extracts of mulberry leaves, respectively. Through the combination of SERS spectra and machine learning algorithms, mulberry leaves were well discriminated with high accuracies in terms of their geographic origins, among which the deep learning algorithm convolutional neural network (CNN) showed the best performance. Taken together, our study established a novel method for predicting the geographic origins of mulberry leaves through the combination of SERS spectra with machine learning algorithms, which strengthened the application potential of the method in the quality evaluation, control and assurance of mulberry leaves.

Effects of UGT1A, CYP2C9/19 and ABAT polymorphisms on plasma concentration of valproic acid in Chinese epilepsy patients.

Aim: To evaluate the association between genetic polymorphisms and plasma concentration-to-dose ratio of valproic acid (CDRV) in Chinese epileptic patients. Methods: A total of 46 epileptic patients treated with valproic acid therapy were enrolled. 18 SNPs in nine genes related to valproic acid were directly sequenced with Sanger methods. Results: Patients carrying UGT1A6 heterozygous genotypes had significantly lower CDRV than those carrying the wild-type genotypes. In contrast, patients with the homozygote genotypes of CYP2C9 and ABAT had higher CDRV than those with the wild-type genotypes and patients with the heterozygous genotypes of CYP2C19 had higher CDRV. Conclusion: Detection of genetic polymorphism in these genes might facilitate an appropriate dose of valproic acid for epileptic patients. Further studies with larger cohorts are necessary to underpin these observations.

Synthesis of Molecularly Imprinted Polymers Based on a New Monomer "2-(4-Vinylphenyl) Quinoline-4-Carboxylic Acid" for the Selective Solid-Phase Extraction of Lamotrigine.

A new molecularly imprinted polymers (MIPs) have been prepared for the high selective extraction of lamotrigine (LTG), a widely used antiepileptic drug, in human serum. The MIPs were polymerized by bulk polymerization using our synthesized compound, 2-(4-vinylphenyl) quinolin-4-carboxylic acid, as functional monomer, which achieved better adsorption specificity than universal MIPs. Then, the molecularly imprinted solid phase extraction (MISPE) based on this material was coupled with high-performance liquid chromatography (HPLC) for the detection of LTG in human serum. The results of method validation showed that the developed method presented a good precision and accuracy, and the linearity was in the range of 1.50-40.00 mg/mL with the limit of quantitation (LOQ) at 0.20 mg/mL. The recovery ranged from 80.8% to 83.8% with RSD ranges from 5.5% to 11.1%. The validated method was successfully used to determine the concentration of LTG in human simulate serum samples.

A simple and easy non-derivatization gas chromatography-mass spectrometry method for simultaneous quantification of valproic acid, gabapentin, pregabalin, and vigabatrin in human plasma.

Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988-0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%-94.5%, 90.0%-90.9%, 90.0%-92.1%, and 88.0%-92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.

Diprenylated flavonoids from licorice induce death of SW480 colorectal cancer cells by promoting autophagy: Activities of lupalbigenin and 6,8-diprenylgenistein.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a well-known herbal medicine, and we previously found that several licorice prenylated flavonoids could cause death of SW480 colorectal cancer cells by promoting autophagy. Given many kinds of prenylated flavonoids in licorice, the activities of other compounds deserve further investigation. In addition, the contribution of isoprenyl groups on the autophagy promotion activities has not been clarified. AIM OF THE STUDY: This study aimed to investigate whether lupalbigenin (LPB) and 6,8-diprenylgenistein (DPG), two licorice diprenylated flavonoids, could induce autophagic cell death of SW480 cells, and clarify the contribution of isoprenyl groups. MATERIALS AND METHODS: Cytotoxic activities of LPB and DPG were tested by using an MTT method, and apoptosis induction effects were evaluated by PI-Annexin V staining-based flow cytometry and protein levels of caspase-3 and PARP-1. Autophagy promotion effects of LPB and DPG were assessed by protein levels of LC3, p62, Akt and mTOR as well as number of autophagosomes in cells, and autophagy inhibitor chloroquine (CQ) was involved to identify the role of autophagy on LPB or DPG-caused death of SW480 cells. In addition, two groups of structurally similar diprenylated, mono-prenylated and free flavonoids were obtained from licorice, which were used to investigate the contribution of isoprenyl groups on their autophagy promotion activities. RESULTS: Both LPB and DPG significantly induced apoptosis of SW480 cells with strong cytotoxic activities, and meanwhile, they also promoted autophagy probably through the Akt/mTOR signaling pathway. Further studies indicated that LPB and DPG could induce autophagic cell death of SW480 cells. Moreover, isoprenyl groups contributed mainly to the cytotoxic and autophagy promotion activities of licorice prenylated flavonoids. CONCLUSION: This study reported for the first time that licorice diprenylated flavonoids LPB and DPG induced death of SW480 cells by promoting autophagy, which was mainly attributed to the isoprenyl groups. The results provided theoretical basis for researches on anti-colorectal cancer drugs and their structural modification.

Development and evaluation of a simple and easy high-performance liquid chromatography-ultraviolet system simultaneously suitable for determination of 24 anti-epileptic drugs in plasma.

We aim to establish a simple and easy high-performance liquid chromatography system coupled with an ultraviolet detector suitable for simultaneous determination of 24 antiepileptic drugs in human plasma. Optimized chromatographic separation was performed on a ZORBAX Eclipse Plus-C18 (4.6 × 150 mm2 , 3.5 µm) column with acetonitrile and 5 mM potassium dihydrogen phosphate water solution as mobile phase. Note that, 24 antiepileptic drugs were divided into three groups and eluted with different gradient procedures, respectively. The column temperature was maintained at 35°C and the detection wavelength was set at 210 nm. Plasma was processed with ethyl acetate or acetonitrile. The calibration curves of 24 antiepileptic drugs demonstrated good linearity within the test range (r > 0.996). The intra- and inter-batch precision and accuracy were all less than 15%, while extraction recoveries were in the range of 74.57-90.89% with the relative standard deviation values less than 15%. The validated methods have been successfully applied to determination of some antiepileptic drugs in rat or patient plasma. Those results indicated that the developed methods were simple and easy, and could be suitable for the determination of 24 antiepileptic drugs in plasma just by changing the gradient elution procedures of mobile phase.

Development of magnetic molecularly imprinted polymers with double templates for the rapid and selective determination of carbamazepine and lamotrigine in serum.

A dual-template magnetic molecularly imprinted polymer (Dt-MMIP) with a specific recognition capability for carbamazepine (CBZ) and lamotrigine (LTG) was synthesized using methacrylic acid as a functional monomer, and ethylene glycol dimethylmethacrylate as a cross-linking agent. A magnetic non-molecularly imprinted polymer without templates (MNIP) was also prepared using the same procedure. The prepared polymers were characterized using scanning electron microscopy, Fourier-transform infrared spectroscopy and adsorption experiments. Results indicated that both Dt-MMIPs and MNIPs were microspherical nanoparticles, and the surface of the Dt-MMIP was rougher than that of the MNIP. In addition, the prepared Dt-MMIPs possessed a higher adsorption capacity and better selectivity for CBZ and LTG than the MNIPs. The maximum static adsorption capacities of Dt-MMIP for CBZ and LTG were 249.5 and 647.9 µg g-1, respectively, whereas those of MNIP were 75.8 and 379.8 µg g-1, respectively. The obtained Dt-MMIPs were applied as a magnetic solid-phase extraction sorbent for the rapid and selective extraction of CBZ and LTG in rat serum samples, and determination was performed by high-performance liquid chromatography with UV detection (HPLC-UV). The developed method of dispersive SPE based on Dt-MMIPs coupled to HPLC-UV has good rapidity and selectivity, and application prospects in serum.

Lipid metabolism disorders and lipid mediator changes of mice in response to long-term exposure to high-fat and high sucrose diets and ameliorative effects of mulberry leaves.

Mulberry leaves exhibit anti-lipogenic and lipid-lowering effects. However, the lipid biomarkers and underlying mechanisms for the improvement of the action of mulberry leaves on obesity and lipid metabolism disorders have not been sufficiently investigated yet. Herein, biochemical analysis combined with metabolomics targeting serum lipid mediators (oxylipins) were used to explore the efficacy and underlying mechanisms of mulberry leaf water extract (MLWE) in high-fat and high-sucrose diet (HFHSD)-fed mice. Our results showed that MLWE supplementation not only decreased body weight gain, serum total triglycerides, low-density lipoprotein cholesterol, alanine transaminase and aspartate transaminase levels, but also increased the serum level of high-density lipoprotein cholesterol. In addition, MLWE supplementation also ameliorated hepatic steatosis and lipid accumulation. These beneficial effects were associated with down-regulating genes involved in oxidative stress, inflammation, and lipogenesis such as acetyl-CoA carboxylase and fatty acid synthase, and up-regulating genes related to lipolysis that encoded peroxisome proliferator-activated receptor α, adiponectin (ADPN), adiponectin receptor (AdipoR) 1, AdipoR2, adenosine monophosphate-activated protein kinase (AMPK) and hormone-sensitive lipase. Moreover, a total of 54 serum lipid mediators were differentially changed in HFHSD-fed mice, among which 11 lipid mediators from n-3 polyunsaturated fatty acids (PUFAs) were apparently reversed by MLWE. These findings indicated that the ADPN/AMPK pathway, anti-inflammation, anti-oxidation, and n-3 PUFA metabolism played important roles in anti-obesity and improvement of lipid metabolism disorders modulated by MLWE supplementation.

Analysis of plasma free amino acids in diabetic rat and the intervention of Ginkgo biloba leaves extract using hydrophilic interaction liquid chromatography coupled with tandem mass-spectrometry.

Amino acids (AAs) are important metabolites that are related with diabetes. However, their roles in the initiation and development of diabetes mellitus (DM), especially in the treatment of Ginkgo biloba leaves extract (GBE) have not been fully explored. Thus, we investigated the roles that AAs played in the progression and GBE supplementation of DM rat induced by streptozotocin. The rats were randomly divided into a normal control group treated with drug-free solution, a normal control group treated with GBE, a DM group treated with drug-free solution, and DM group treated with GBE; and maintained on this protocol for 9 weeks. Rat plasma was collected from the sixth week to the ninth week and then analyzed with the optimized hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method. A total of 17 AAs with differential levels were monitored to indicate dysfunction of AAs metabolism to confirm the occurrence and development of DM. Treatment with GBE partially reversed the changes seen in seven AAs including leucine, isoleucine, tyrosine, glutamic acid, asparagines, lysine and alanine in DM rats, indicating that GBE could prevent the occurrence and development of DM by acting on AAs metabolism. The improvement of those AAs metabolism disorders may play a considerable role in the treatment of GBE on the occurrence and development of DM. Those findings potentially promote the understanding of the pathogenic progression of DM and reveal the therapeutic mechanism of GBE against DM.

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.

Chemical Antioxidant Quality Markers of Chrysanthemum morifolium Using a Spectrum-Effect Approach.

Traditionally, the quality evaluation of Chrysanthemum morifolium (CM) cv. (Juhua) attributes its habitats and processing methods, however, this strategy of neglecting bioactive ingredients usually results in deviation of quality evaluation. This study aims to explore the quality marker (Q-marker) based on spectrum-effect relationship and quality control strategy of CMs. The chromatographic fingerprint of 30 flower head samples of CMs from five different habitats including Hang-baiju, Gongju, Huaiju, Taiju and Boju were constructed by high performance liquid chromatography and analyzed through chemometrics methods such as similarity analysis (SA), cluster analysis (CA) and principal component analysis (PCA). The common peaks were quantified by external standard method and relative correction factor method. The in-vitro radical scavenging capacity assays of DPPH·, ·OH and ABTS were carried out. The Q-marker was explored by the correlation analysis between the contents of common peaks and in-vitro radical scavenging capacity, and then used to evaluate the quality of 30 flower head samples of CMs. A total of eight common peaks were appointed in 30 flower head samples of CMs, and their similarities ranged from 0.640 to 0.956. CA results showed that 30 flower head samples of CMs could be divided into five categories with reference to the Euclidean distance of 5. PCA results showed that common peaks played a major role in differential contribution of CMs. The quantification of common peaks hinted that their contents possessed significant variation whether for different accessions or the same accessions of CMs. The correlation analysis showed that chlorogenic acid, 3,5-O-dicaffeoylquinic acid, unknown peak 1, 4,5-O-dicaffeoylquinic acid and kaempferol-3-O-rutinoside could be used as the Q-markers for the quality evaluation of 30 flower head samples of commercially available CMs. The analysis strategy that combines chromatographic fingerprint analysis, multiple ingredients quantification, in-vitro chemical anti-oxidant activity evaluation and spectrum-effect relationship analysis clarified the therapeutic material basis and discovered the Q-markers, which possibly offers a more comprehensive quality assessment of CMs.

Prenylated phenolic compounds from licorice (Glycyrrhiza uralensis) and their anti-inflammatory activity against osteoarthritis.

Osteoarthritis is a significant driver of disability in the elderly with increasing prevalence, and inflammation plays a vital role on its etiology. Licorice is commonly used as a traditional Chinese medicine or food additive, and its prenylated phenolic compounds were recently reported to be able to inhibit osteoarthritis with anti-inflammatory activity. In order to explore more anti-osteoarthritic prenylated phenolic compounds from licorice, we isolated ten compounds (1-10), with three new ones (1-3), from the ethyl acetate extract of Glycyrrhiza uralensis. Compound 2 (glycyuralin R) was a racemic 3-phenoxy-chromanone, and we achieved its chiral separation for the first time. Compounds 1, 2, 7 and 8 showed significant NO inhibitory ability in IL-1ß-stimulated mouse primary chondrocytes, and we further confirmed the anti-inflammatory activity of 1 (glycyuralin Q) by evaluating its effect on osteoarthritis-related iNOS, COX-2, TNF-α, IL-6, MMP3, MMP13 and NF-κB based on various experimental methods. These results clarified the potential of several prenylated phenolic compounds, especially 1 in licorice, as the lead compounds for osteoarthritis.

Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes.

BACKGROUND: Protection of pancreatic islet cells against dysfunction or death by regulating autophagy is considered to be an effective method for treatment of type 2 diabetes mellitus (T2DM). Morus alba leaves (mulberry leaves), a popular herbal medicine, have been used for prevention of T2DM since ancient times. PURPOSE: This study aimed to clarify whether Morus alba leaves ethanol extract (MLE) could protect islet cells in vivo and in vitro by regulating autophagy in T2DM, and explore the possible mechanism of action. METHODS: The main chemical constituents in MLE were analyzed by HPLC. The T2DM rat model was induced via high-fat diet combined with peritoneal injection of low-dose streptozotocin, and MLE was administered by oral gavage. Fasting blood glucose (FBG) and plasma insulin were measured, and homeostatic model assessment of ß cell function (HOMA-ß) and insulin resistance (HOMA-IR) were determined. The histomorphology of pancreas islets was evaluated by haematoxylin and eosin staining. In palmitic acid (PA)-stressed INS-1 rat insulinoma cells, cell viability was assayed by an MTT method. Expression of the autophagy-related proteins LC3 I/II, p62, p-AMPK and p-mTOR in islet tissues and INS-1 cells was evaluated by western blotting or immunohistochemistry analysis. RESULTS: The four main chemical constituents in MLE were identified as chlorogenic acid, rutin, isoquercitrin and quercitrin. MLE ameliorated hyperglycemia, insulin resistance and dyslipidemia of T2DM rats with prominent therapeutic effect. Further study indicated that MLE observably improved islet function, alleviated islet injury of T2DM rats, and inhibited PA-induced INS-1 cell death. On the other hand, MLE significantly induced autophagy in islet cells both in vivo and in vitro, and autophagy inhibitors abolished its therapeutic effect on T2DM rats and protective effect on islet cells. Apart from this, MLE markedly activated the AMPK/mTOR pathway in INS-1 cells, and the AMPK inhibitor prevented the autophagy induction ability of MLE. CONCLUSION: Together, MLE could protect islet cells against dysfunction and death by inducing AMPK/mTOR-mediated autophagy in T2DM, and these findings provide a new perspective for understanding the treatment mechanism of Morus alba leaves against T2DM.

Enrichment and Quantitative Determination of Free 3,5- Diiodothyronine, 3',5'-Diiodothyronine, and 3,5-Diiodothyronamine in Human Serum of Thyroid Cancer by Covalent Organic Hyper Cross-linked Poly-ionic Liquid.

The incidence of thyroid cancer is increasing worldwide. So far, still no non-invasive clinical test biomarkers were developed for the diagnosis of thyroid cancer. The diiodothyronines (T2s) are precursors and metabolites of thyroid hormone (T4). Some reports predict that T2s may be associated with several thyroid diseases, especially the thyroid cancer. Detecting free T2s in human serum may help the diagnosis of thyroid cancer. However, few works have reported the detection of T2s due to their trace amounts. Here we developed a novel hyper organic cross-linked poly ionic liquid (PIL) material for the enrichment of three main compounds in T2s family, including 3,5- diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,5-diiodothyronamine (3,5-T2AM). This PIL material provided specific enrichment superiority for three T2s. After enrichment, the signal intensity of 3,5-T2, 3',5'-T2, and 3,5-T2AM increased 14, 132 and 1.6 folds, respectively, with LOQ of 76, 87, and 107 fM, respectively. Finally, we successfully applied PIL material coupled with HPLC-ESI-MS/MS in enrichment and quantitative determination of free 3,5-T2, 3',5'-T2, and 3,5-T2AM in human serum of 45 thyroid cancer patients and 15 healthy people. We also used free thyroid hormone (FT4) as the calibration reference to eliminate individual differences. We found that the levels of 3,5-T2 (P < 0.001), and 3',5'-T2 (P = 0.001) in patients with thyroid cancer were significantly higher than those in healthy people. Additionally, we further investigated the power of different T2 thyroid hormones divided FT4 to classify thyroid cancer patients and healthy people. And 3,5-T2/FT4 had the highest classification performance for discriminating thyroid cancer patients from healthy people at certain threshold, indicating that 3,5-T2/FT4 in human serum can act as potential biomarkers for "non-invasive" clinical diagnosis of thyroid cancer.