Unified framework for brain connectivity-based biomarkers in neurodegenerative disorders.

Background: Brain connectivity is useful for deciphering complex brain dynamics controlling interregional communication. Identifying specific brain phenomena based on brain connectivity and quantifying their levels can help explain or diagnose neurodegenerative disorders. Objective: This study aimed to establish a unified framework to identify brain connectivity-based biomarkers associated with disease progression and summarize them into a single numerical value, with consideration for connectivity-specific structural attributes. Methods: This study established a framework that unifies the processes of identifying a brain connectivity-based biomarker and mapping its abnormality level into a single numerical value, called a biomarker abnormality summarized from the identified connectivity (BASIC) score. A connectivity-based biomarker was extracted in the form of a connected component associated with disease progression. BASIC scores were constructed to maximize Kendall's rank correlation with the disease, considering the spatial autocorrelation between adjacent edges. Using functional connectivity networks, we validated the BASIC scores in various scenarios. Results: Our proposed framework was successfully applied to construct connectivity-based biomarker scores associated with disease progression, characterized by two, three, and five stages of Alzheimer's disease, and reflected the continuity of brain alterations as the diseases advanced. The BASIC scores were not only sensitive to disease progression, but also specific to the trajectory of a particular disease. Moreover, this framework can be utilized when disease stages are measured on continuous scales, resulting in a notable prediction performance when applied to the prediction of the disease. Conclusion: Our unified framework provides a method to identify brain connectivity-based biomarkers and continuity-reflecting BASIC scores that are sensitive and specific to disease progression.

On the reliability of deep learning-based classification for Alzheimer's disease: Multi-cohorts, multi-vendors, multi-protocols, and head-to-head validation.

Structural changes in the brain due to Alzheimer's disease dementia (ADD) can be observed through brain T1-weighted magnetic resonance imaging (MRI) images. Many ADD diagnostic studies using brain MRI images have been conducted with machine-learning and deep-learning models. Although reliability is a key in clinical application and applicability of low-resolution MRI (LRMRI) is a key to broad clinical application, both are not sufficiently studied in the deep-learning area. In this study, we developed a 2-dimensional convolutional neural network-based classification model by adopting several methods, such as using instance normalization layer, Mixup, and sharpness aware minimization. To train the model, MRI images from 2,765 cognitively normal individuals and 1,192 patients with ADD from the Samsung medical center cohort were exploited. To assess the reliability of our classification model, we designed external validation in multiple scenarios: (1) multi-cohort validation using four additional cohort datasets including more than 30 different centers in multiple countries, (2) multi-vendor validation using three different MRI vendor subgroups, (3) LRMRI image validation, and finally, (4) head-to-head validation using ten pairs of MRI images from ten individual subjects scanned in two different centers. For multi-cohort validation, we used the MRI images from 739 subjects from the Alzheimer's Disease Neuroimaging Initiative cohort, 125 subjects from the Dementia Platform of Korea cohort, 234 subjects from the Premier cohort, and 139 subjects from the Gachon University Gil Medical Center. We further assessed classification performance across different vendors and protocols for each dataset. We achieved a mean AUC and classification accuracy of 0.9868 and 0.9482 in 5-fold cross-validation. In external validation, we obtained a comparable AUC of 0.9396 and classification accuracy of 0.8757 to other cross-validation studies in the ADNI cohorts. Furthermore, we observed the possibility of broad clinical application through LRMRI image validation by achieving a mean AUC and classification accuracy of 0.9404 and 0.8765 at cross-validation and AUC and classification accuracy of 0.8749 and 0.8281 at the ADNI cohort external validation.

Metabolomic Profiling of Citrus unshiu during Different Stages of Fruit Development.

Citrus fruits undergo significant metabolic profile changes during their development process. However, limited information is available on the changes in the metabolites of Citrus unshiu during fruit development. Here, we analyzed the total phenolic content (TPC), total carotenoid content (TCC), antioxidant activity, and metabolite profiles in C. unshiu fruit flesh during different stages of fruit development and evaluated their correlations. The TPC and antioxidant activity significantly decreased during fruit development, whereas the TCC increased. The metabolite profiles, including sugars, acidic compounds, amino acids, flavonoids, limonoids, carotenoids, and volatile compounds (mono- and sesquiterpenes), in C. unshiu fruit flesh also changed significantly, and a citrus metabolomic pathway related to fruit development was proposed. Based on the data, C. unshiu fruit development was classified into three groups: Group 1 (Aug. 1), Group 2 (Aug. 31 and Sep. 14), and Group 3 (Oct. 15 and Nov. 16). Although citrus peel was not analyzed and the sensory and functional qualities during fruit development were not investigated, the results of this study will help in our understanding of the changes in chemical profile during citrus fruit development. This can provide vital information for various applications in the C. unshiu industry.

A Significant Change in Free Amino Acids of Soybean (Glycine max L. Merr) through Ethylene Application.

In this study, the changes in free amino acids of soybean leaves after ethylene application were characterized based on quantitative and metabolomic analyses. All essential and nonessential amino acids in soybean leaves were enhanced by fivefold (250 to 1284 mg/100 g) and sixfold (544 to 3478 mg/100 g), respectively, via ethylene application. In particular, it was found that asparagine is the main component, comprising approximately 41% of the total amino acids with a twenty-five fold increase (78 to 1971 mg/100 g). Moreover, arginine and branched chain amino acids (Val, Leu, and Ile) increased by about 14 and 2-5 times, respectively. The increase in free amino acid in stem was also similar to the leaves. The metabolites in treated and untreated soybean leaves were systematically identified by gas chromatography-mass spectrometry (GC-MS), and partial variance discriminant analysis (PLS-DA) scores and heat map analysis were given to understand the changes of each metabolite. The application of ethylene may provide good nutrient potential for soybean leaves.

Metabolite profiling reveals organ-specific flavone accumulation in Scutellaria and identifies a scutellarin isomer isoscutellarein 8-O-ß-glucuronopyranoside.

Scutellaria is a genus of plants containing multiple species with well-documented medicinal effects. S. baicalensis and S. barbata are among the best-studied Scutellaria species, and previous works have established flavones to be the primary source of their bioactivity. Recent genomic and biochemical studies with S. baicalensis and S. barbata have advanced our understanding of flavone biosynthesis in Scutellaria. However, as over several hundreds of Scutellaria species occur throughout the world, flavone biosynthesis in most species remains poorly understood. In this study, we analyzed organ-specific flavone profiles of seven Scutellaria species, including S. baicalensis, S. barbata, and two species native to the Americas (S. wrightii to Texas and S. racemosa to Central and South America). We found that the roots of almost all these species produce only 4'-deoxyflavones, while 4'-hydroxyflavones are accumulated exclusively in their aerial parts. On the other hand, S. racemosa and S. wrightii also accumulated high levels of 4'-deoxyflavones in their aerial parts, different with the flavone profiles of S. baicalensis and S. barbata. Furthermore, our metabolomics and NMR study identified the accumulation of isoscutellarein 8-O-ß-glucuronopyranoside, a rare 4'-hydroxyflavone, in the stems and leaves of several Scutellaria species including S. baicalensis and S. barbata, but not in S. racemosa and S. wrightii. Distinctive organ-specific metabolite profiles among Scutellaria species indicate the selectivity and diverse physiological roles of flavones.

Aldoxime Metabolism Is Linked to Phenylpropanoid Production in Camelina sativa.

Plants produce diverse secondary metabolites. Although each metabolite is made through its respective biosynthetic pathway, plants coordinate multiple biosynthetic pathways simultaneously. One example is an interaction between glucosinolate and phenylpropanoid pathways in Arabidopsis thaliana. Glucosinolates are defense compounds made primarily from methionine and tryptophan, while phenylpropanoids are made from phenylalanine. Recent studies have shown that the accumulation of glucosinolate intermediate such as indole-3-acetaldoxime (IAOx) or its derivatives represses phenylpropanoid production via the degradation of phenylalanine ammonia lyase (PAL) functioning at the entry point of the phenylpropanoid pathway. Given that IAOx is a precursor of other bioactive compounds other than glucosinolates and that the phenylpropanoid pathway is present in most plants, we hypothesized that this interaction is relevant to other species. Camelina sativa is an oil crop and produces camalexin from IAOx. We enhanced IAOx production in Camelina by overexpressing Arabidopsis CYP79B2 which encodes an IAOx-producing enzyme. The overexpression of AtCYP79B2 results in increased auxin content and its associated morphological phenotypes in Camelina but indole glucosinolates were not detected in Camelina wild type as well as the overexpression lines. However, phenylpropanoid contents were reduced in AtCYP79B2 overexpression lines suggesting a link between aldoxime metabolism and phenylpropanoid production. Interestingly, the expression of PALs was not affected in the overexpression lines although PAL activity was reduced. To test if PAL degradation is involved in the crosstalk, we identified F-box genes functioning in PAL degradation through a phylogenetic study. A total of 459 transcript models encoding kelch-motifs were identified from the Camelina sativa database. Among them, the expression of CsKFBs involved in PAL degradation is up-regulated in the transgenic lines. Our results suggest a link between aldoxime metabolism and phenylpropanoid production in Camelina and that the molecular mechanism behind the crosstalk is conserved in Arabidopsis and Camelina.

Highly potent bacterial neuraminidase inhibitors, chromenone derivatives from Flemingia philippinensis.

The chromenone derivatives (1-4) from the root part of Flemingia philippinensis showed a significant inhibition against bacterial neuraminidase (NA) which plays a pivotal role in a cellular interaction including pathogenesis of bacterial infection and subsequent inflammation. The compounds 1 and 2 were the new compounds, philippin D (1) and philippin E (2). In particular, compounds (1-3) exhibited sub micromolar levels of IC50 values with 0.75, 0.54, and 0.07 µM. This is the first report that chromenone skeleton emerged as a lead structure of bacterial NA inhibition. In kinetic study, 8,8-diprenyl compounds displayed competitive inhibitory mode, whereas 4a,8-diprenyl ones showed noncompetitive behavior. It was manifested that all competitive inhibitors (1 and 2) were simple reversible slow-binding against bacterial NA. The binding affinities (KSV) of inhibitors to enzyme were agreement with their respective inhibitory potencies. Molecular docking data confirmed that the position of 3-methyl-2-butenyl substituent affects inhibitory mechanism against CpNanI. The tri-arginyl cluster of R266, R555, and R615 and D291 in NanI tightly interact with the competitive inhibitors.

Phenylephrine, a small molecule, inhibits pectin methylesterases.

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1 µM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.

Improvement of nutritional components and in vitro antioxidative properties of soy-powder yogurts using Lactobacillus plantarum.

This research was the first to demonstrate changes in nutritional compositions (isoflavone and CLA) from the 50% methanol extracts of soy-powder milk (SPM) and soy-powder yogurt (SPY) through fermentation using Lactobacillus plantarum S48 and P1201 strains. The radical scavenging activities and protective effects against oxidative stress in LLC-PK1 cells were also investigated. The average physicochemical characteristics including acidity and viable cell number as well as ß-glucosidase activity increased with 0.2 â†’ 0.7%, 7.5 â†’ 9.8 log cfu/mL, and 0.0 3 â†’ 1.75 U/g in SPYs. Total average isoflavones were considerably reduced (3180.3 â†’ 2018.3 µg/g) with the increase of aglycone contents (191.8 â†’ 770.2 µg/g), especially, daidzein exhibited the most remarkable increase rate (98.6 â†’ 460.9 µg/g; > 4.8 times) during fermentation. The CLA and total phenolics also increased with significant differences (ND → 1.6 mg/g; 2.4 â†’ 3.6 mg/GAE/g) between SPM and SPY. Interestingly, the cis-9, trans-11 CLA showed approximately 90% in total content. Moreover, the scavenging capacities against three radicals markedly increased with about 30% in SPYs, as the following order: ABTS > hydroxyl > DPPH. The protective effects on oxidative stress (pyrogallol: O2-, SNP: NO, and SIN-1: ONOO-) were also observed high cell viabilities (>10%) under LLC-PK1 cellular system. Our results suggest that SPY may be utilized as a potent source regarding natural antioxidants and beneficial components for health food and medical uses.

Antioxidant Activities of Phenolic Metabolites from Flemingia philippinensis Merr. et Rolfe and Their Application to DNA Damage Protection.

F. philippinensis Merr. et Rolfe has been cultivated on a large scale and is widely consumed by local inhabitants as an important nutraceutical, especially against rheumatism which has a deep connection with antioxidants. In this study, a total of 18 different phenolic metabolite compounds in F. philippinensis were isolated and identified, and evaluated for their antioxidant and DNA damage protection potential. The antioxidant activity of the 18 identified compounds was screened using DPPH, ORAC, hydroxyl and superoxide radical scavenging assays. The antioxidant potential of the compounds was found to differ by functionality and skeleton. However, most compounds showed a good antioxidant potential. In particular, seven of the identified compounds, namely, compounds 2, 3, 6, 10, 11, 15 and 16, showed significant protective effects on pBR322 plasmid DNA against the mutagenic and toxic effects of Fenton's reaction. The most active compound, compound 2, displayed a dose-dependent DNA damage protection potential in the range of 7.5~60.0 µM. The DNA damage protective effect of the identified compounds was significantly correlated with the hydroxyl radical scavenging activity. Compounds that exhibited effective (IC50 = 5.4~12.5 µg/mL) hydroxyl radical scavenging activity were found to be the ones with higher DNA damage protection potential.

Competitive neutrophil elastase inhibitory isoflavones from the roots of Flemingia philippinensis.

Flemingia philippinensis has been used throughout history to cure rheumatism associated with neutrophil elastase (NE). In this study, we isolated sixteen NE inhibitory flavonoids (1-16), including the most potent and abundant prenyl isoflavones (1-9), from the F. philippinensis plant. These prenyl isoflavones (2, 3, 5, 7, and 9) competitively inhibited NE, with IC50 values of 1.3-12.0 µM. In addition, they were reversible, simple, slow-binding inhibitors according to their respective parameters. Representative compound 3 had an IC50 = 1.3 µM, k3 = 0.04172 µM-1 min-1, k4 = 0.0064 min-1, and Kiapp = 0.1534 µM. The Kik/Kiv ratios (18.5 ∼ 24.6) for compound 3 were consistent with typical competitive inhibitors. The prenyl functionality of isoflavones significantly affected inhibitory potencies and mechanistic behavior by shifting the competitive mode to a noncompetitive one. The remaining flavonoids (10-16) were confirmed as mixed type I inhibitors that preferred to bind free enzyme rather than the enzyme-substrate complex. Fluorescence quenching analyses indicated that the inhibitory potency (IC50) closely followed the binding affinity (KSV).

Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense.

Four new caged xanthones (1-4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1-6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1-6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC50 values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining Km, Vmax, and the ratio of Kik and Kiv, which revealed that all the compounds behaved as competitive inhibitors.

Isolation and Characterization of Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitory Polyphenolic Compounds From Dodonaea viscosa and Their Kinetic Analysis.

Diabetes mellitus is one of a major worldwide concerns, regulated by either defects in secretion or action of insulin, or both. Insulin signaling down-regulation has been related with over activity of protein tyrosine phosphatase 1B (PTP1B) enzyme, which has been a promising target for the treatment of diabetes mellitus. Herein, activity guided separation of methanol extract (95%) of Dodonaea viscosa aerial parts afforded nine (1-9) polyphenolic compounds, all of them were identified through spectroscopic data including 2D NMR and HREIMS. Subsequently, their PTP1B inhibitory potentials were evaluated, in which all of the isolates exhibited significant dose-dependent inhibition with IC50 13.5-57.9 µM. Among them, viscosol (4) was found to be the most potent compound having IC50 13.5 µM. In order to unveil the mechanistic behavior, detailed kinetic study was carried out, in which compound 4 was observed as a reversible, and mixed type I inhibitor of PTP1B with inhibitory constant (Ki) value of 4.6 µM. Furthermore, we annotated the major metabolites through HPLC-DAD-ESI/MS analysis, in which compounds 3, 6, 7, and 9 were found to be the most abundant metabolites in D. viscosa extract.

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization.

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1-12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC50s of (2.4-52.5 µM), and α-glucosidase with IC50 values of (1.7-72.7 µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC50 = 2.4 µM for 3, 2.8 µM for 7) and α-glucosidase (IC50 = 4.8 µM for 3, 1.7 µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: Kiapp = 2.4 µM; k5 = 0.05001 µM-1 S-1 and k6 = 0.02076 µM-1 S-1.

Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa.

Protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase are important targets to treat obesity and diabetes, due to their deep correlation with insulin and leptin signalling, and glucose regulation. The methanol extract of Paulownia tomentosa fruits showed potent inhibition against both enzymes. Purification of this extract led to eight geranylated flavonoids (1-8) displaying dual inhibition of PTP1B and α-glucosidase. The isolated compounds were identified as flavanones (1-5) and dihydroflavonols (6-8). Inhibitory potencies of these compounds varied accordingly, but most of the compounds were highly effective against PTP1B (IC50 = 1.9-8.2 µM) than α-glucosidase (IC50 = 2.2-78.9 µM). Mimulone (1) was the most effective against PTP1B with IC50 = 1.9 µM, whereas 6-geranyl-3,3',5,5',7-pentahydroxy-4'-methoxyflavane (8) displayed potent inhibition against α-glucosidase (IC50 = 2.2 µM). All inhibitors showed mixed type Ι inhibition toward PTP1B, and were noncompetitive inhibitors of α-glucosidase. This mixed type behavior against PTP1B was fully demonstrated by showing a decrease in Vmax, an increase of Km, and Kik/Kiv ratio ranging between 2.66 and 3.69.

Phytochemical profile and angiotensin I converting enzyme (ACE) inhibitory activity of Limonium michelsonii Lincz.

Members of the genus Limonium are widely used as medicinal herbs due to their health-promoting effects, such as an ability to improve blood circulation by inhibiting angiotensin I converting enzyme (ACE). While the potential of L. michelsonii Lincz. (a medicinal plant endemic to Kazakhstan) to inhibit ACE has been demonstrated, the inhibitory activities of its secondary metabolites have not been explored. In this work, the principal phenolic compounds (1-20) among these metabolites were isolated to determine the components responsible for ACE inhibition. The natural abundances of the active constituents within the target plant were characterized by UPLC-Q-TOF/MS analysis. All of the isolated compounds except for gallates 10-12 were found to significantly inhibit ACE, with IC50 values of between 7.1 and 138.4 µM. Unexpectedly, the flavonol glycosides 16-20 were observed to be more potent than the corresponding aglycones 4 and 5. For example, quercetin (4) had IC50 = 30.3 µM, whereas its glycosides (16, 17) had IC50 = 10.2 and 14.5 µM, respectively. A similar trend was observed for myricetin (5) and its glycosides (18-20). In a kinetic study, the flavonols 3-5 and 16-20 and the dihydroflavonols 8 and 9 behaved as competitive inhibitors, whereas other flavones (1, 2, 13-15) and flavanones (6, 7) performed noncompetitive inhibition.

Competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors, prenylated caged xanthones from Garcinia hanburyi and their inhibitory mechanism.

Protein tyrosine phosphatase 1B (PTP1B) plays important role in diabetes, obesity and cancer. The methanol extract of the gum resin of Garcinia hanburyi (G. hanburyi) showed potent PTP1B inhibition at 10µg/ml. The active compounds were identified as prenylated caged xanthones (1-9) which inhibited PTP1B in dose-dependent manner. Carboxybutenyl group within caged motif (A ring) was found to play a critical role in enzyme inhibition such as 1-6 (IC50s=0.47-4.69µM), whereas compounds having hydroxymethylbutenyl 7 (IC50=70.25µM) and methylbutenyl 8 (IC50>200µM) showed less activity. The most potent inhibitor, gambogic acid 1 (IC50=0.47µM) showed 30-fold more potency than ursolic acid (IC50=15.5µM), a positive control. In kinetic study, all isolated xanthones behaved as competitive inhibitors which were fully demonstrated with Km, Vmax and Kik/Kiv ratio. It was also proved that inhibitor 1 operated under the enzyme isomerization model having k5=0.0751µM-1S-1, k6=0.0249µM-1S-1 and Kiapp=0.499µM. To develop a pharmacophore model, we explored the binding sites of compound 1 and 7 in PTP1B. These modeling results were in agreement with our findings, which revealed that the inhibitory activities are tightly related to caged motif and prenyl group in A ring.

Ethylene Induced a High Accumulation of Dietary Isoflavones and Expression of Isoflavonoid Biosynthetic Genes in Soybean (Glycine max) Leaves.

Dietary isoflavones, daidzein and genistein are of huge interest in the nutraceutical field due to their practical application to postmenopause complications. This study is the first report an efficient method to prepare isoflavone rich soybean leaves (soyleaves) which is an edible food stuff in Asian countries. The preharvest treatment of ethylene highly stimulated the level of isoflavone in soyleaves. Annotation and quantification of metabolites were determined by UPLC-Q-TOF-MS and HPLC. Phenolic metabolites of soyleaves are mostly kaempferol glycosides, but not dietary isoflavones. The accumulated isoflavones by ethylene treatment were determined to be daidzin 1, genistin 2, malonyldaidzin 3 and malonylgenistin 4, which were easily hydrolyzed to daidzein and genistein by ß-glucosidase. Total content of dietary isoflavones was increased up to 13854 µg/g. The most suitable condition was estimated to be 250 µg/g ethylene or 200 µg/g ethephon (ethylene donor) treatment at the R3 growth stage. The ratio of daidzein and genistein glycosides was approximately 5 to 3. The accumulated isoflavonoid biosynthesis pathway genes were identified within the transcriptome of soyleaves tissues at 1 day after treatment of ethephon. The quantitative RT-PCR analysis of these genes indicated significantly higher expression of CHS, CHI, IFS, HID, IF7GT, and IF7MaT compared to control leaves. These findings suggest that ethylene activates a set of structural genes involved in isoflavonoid biosynthesis, thereby leading to enhanced production of isoflavones in soybean plants.

Potent bacterial neuraminidase inhibitors, anthraquinone glucosides from Polygonum cuspidatum and their inhibitory mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: P. cuspidatum is a popular Chinese medicinal herb, having a long history of usage in traditional Chinese medicine for the treatment of several inflammatory diseases in the form of powders and decoctions. Similarly there are many reports that P. cuspidatum has antibacterial and anti-inflammatory effects, both of which are properties associated with compounds having activity against bacterial neuraminidase (BNA). AIM OF THE STUDY: We investigated whether P. cuspidatum's metabolites exhibited BNA inhibition. Consistent with our hypothesis, we found several inhibitors from the methanol extract of this plant, and then fully characterized their inhibitory mechanisms. MATERIALS AND METHODS: Activity guided separation of methanol extract led to isolation of individual constituents, and subsequently their structures were elucidated by spectroscopic analysis. Detailed kinetic behaviors of BNA inhibitors were explored by showing the changes of Km and Vmax, the ratios of KI/KIS and Kik/Kiv, and fluorescence quenching effect. RESULTS AND CONCLUSION: This study attempted to isolate the responsible metabolites and elucidate the BNA inhibitory mechanism. The principal BNA inhibitory compounds (2-6) were identified as emodin (2), physcion-8-O-ß-D-glucopyranoside (3), emodin-8-O-ß-D-glucopyranoside (4), emodin-1-O-ß-D-glucopyranoside (5), and 2-methoxy-6-acetyl-7-methyljuglone (6). Unexpectedly, anthraquinone glucosides (3-5) were much more potent than their corresponding aglycones (1 and 2). For example, emodin (2) had an IC50=5.4µM, whereas its glucosides (4 and 5) had IC50=0.85µM and 0.43µM respectively. A similar trend was observed with physcion (1, IC50>200µM) and its glucoside (3, IC50=6.2µM). The anthraquinone (2) was mixed type I inhibitor, whereas its glucosides (4 and 5) were noncompetitive. In addition, the fluorescence quenching study showed that the affinity constants (KSV) of inhibitors increased in proportion to their inhibitory potencies. Furthermore, we quantified the major and minor metabolites through UPLC-PDA-Q-TOF/MS, and revealed that the most potent inhibitors were the major constituents. This result contributes to our understanding of P. cuspidatum utility as functional food stuff and widely used herbal medicine.