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BackgroundBrain 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.ObjectiveThis 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.MethodsThis 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.ResultsOur 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.ConclusionOur unified framework provides a method to identify brain connectivity-based biomarkers and continuity-reflecting BASIC scores that are sensitive and specific to disease progression.

Citrus fruits undergo significant metabolic profile changes during their development process. However, limited information is available on the changes in the metabolites of

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-β-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.

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.

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.

Activity-guided phytochemical investigation of the methanol extract of Usnea longissima led to the isolation of nine (1–9) phenolic metabolites, including compounds 6 and 7, which were reported for the first time from this species. All isolated compounds were completely characterized through spectroscopic analysis, including 2D NMR and HREIMS data. Individually, the activity of each purified compound was assessed against bacterial neuraminidase (BNA), and most of them displayed dose-dependent inhibition. Among the purified compounds, compounds 2 and 3 exhibited significant inhibitory activities, with IC50 values of 7.8 and 8.2 μM, respectively, and had three-fold greater potency than the reference compound quercetin (IC50 = 21.4 μM). Furthermore, enzyme kinetics revealed that compounds 2 and 3 exhibited reversible and mixed type-I inhibitory behaviors, with inhibitory constant (Ki) values of 6.8 and 7.2 μM, respectively. Our results revealed the potential of U. longissima in inhibiting the enzyme BNA and highlighted its additional medicinal properties.

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.

This study evaluated the changes in γ-aminobutyric acid (GABA) and isoflavone aglycone contents from soy powder yogurt (SPY) due to sprouting of soybean (1 cm) and fermentation with Lactobacillus brevis. The levels of GABA and the aglycone form increased, and the glutamate decarboxylase and β-glucosidase activities increased; however, the isoflavone glycoside and malonylglycoside contents decreased after fermentation for 72 h. In particular, after 60 h, the SPY presented the highest GABA content (120.38 mg/100 mL). The highest daidzein (179.93 µg/g), glycitein (44.10 µg/g), and genistein (126.24 µg/g) contents were present after 72 h of fermentation. In addition, the 2,2-diphenyl-1-picrylhydrazyl, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and hydroxyl radical scavenging activities increased from 69.65, 97.94, and 70.90% during this fermentation, respectively. This result suggests that SPY may be used for the preparation of high-protein soybean with high GABA and isoflavone aglycone contents, which can then be used as a natural ingredient of functional foods.

A series of flavonoids were isolated from Dodonaea Viscosa and tested for inhibition of human neutrophil elastase (HNE), enzyme involved in inflammatory disorders. Isolated compounds were identified as a novel flavonol ( 1 ) along with eight known flavonoids ( 2 – 9 ). Novel flavonol, visconata ( 1 ) has a very rare skeleton having odd numbered long chain (C19) fatty acid, which was completely identified by mass fragmentation and 2D NMR analysis. All compounds ( 1 – 9 ) inhibited HNE in dose dependent manner with IC 50 s ranging between 2.4 and 150 μM. Visconata ( 1 ) emerged to be the most potent compound with 2.4 μM of IC 50 . In kinetic studies, compound ( 1 ) was observed to be reversible, noncompetitive inhibitor having K i = 1.8 μM, whereas other flavonoids ( 2 – 9 ) displayed mixed type inhibition.

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.

This study evaluated the changes of physiochemical properties, phytochemical compounds (isoflavones and phenolic acids), and biological activity during the fermentation of Doenjang without and with bitter melon powder (BMP). The pH decreased from 6.41-5.83 to 5.81-5.24, during the fermentation of Doenjang, while the acidity increased from 0.42-0.65% to 1.28-1.48%. The viable cell numbers of Bacillus and Yeast, salinity, and total amino acid contents increased at the end fermentation (60 day). Also, the fermented Doenjang (FD) with 10% BMP showed the highest γ-aminobutyric acid (GABA, 129.87 mg/100 g) contents, among all the Doenjang samples. The FD exhibited significantly higher inhibitory activities than unfermented Doenjang (UFD) on radicals and α-glucosidase. The phytochemical compounds including isoflavone-aglycones and phenolic acids increased, whereas isoflavoneglycosides decreased in the BM following fermentative processing. Moreover, the total phenolic, isoflavone-aglycone, and phenolic acid contents were markedly increased, leading to a general increase in antioxidant and α-glucosidase inhibition activities after fermentation. These results suggest that BMP may be used to prepare a new type of fermented Doenjang with improved antioxidant and antidiabetic activities.

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.

The α-glucosidase inhibitory potential of Tribulus terrestris extracts has been reported but as yet the active ingredients are unknown. This study attempted to isolate the responsible metabolites and elucidate their inhibition mechanism of α-glucosidase. By fractionating T. terristris extracts, three cinnamic acid amide derivatives (1–3) were ascertained to be active components against α-glucosidase. The lead structure, N-trans-coumaroyltyramine 1, showed significant inhibition of α-glucosidase (IC50 = 0.42 μM). Moreover, all active compounds displayed uncompetitive inhibition mechanisms that have rarely been reported for α-glucosidase inhibitors. This kinetic behavior was fully demonstrated by showing a decrease of both Km and Vmax, and Kik/Kiv ratio ranging between 1.029 and 1.053. We progressed to study how chemical modifications to the lead structure 1 may impact inhibition. An α, β-unsaturation carbonyl group and hydroxyl group in A-ring of cinnamic acid amide emerged to be critical functionalities for α-glucosidase inhibition. The molecular modeling study revealed that the inhibitory activities are tightly related to π-π interaction as well as hydrogen bond interaction between enzyme and inhibitors.

Protein tyrosine phosphatase 1B (PTP1B) is an important target to treat obesity and diabetes due to its key roles in insulin and leptin signaling. The MeOH extracts of the root bark of Flemingia philippinensis yielded eight inhibitory molecules (1-8) capable of targeting PTP1B. Three of them were identified to be novel compounds, philippin A (1), philippin B (2), and philippin C (3) which have a rare 3-phenylpropanoyl chromenedione skeleton. The other compounds (4-8) were known prenylated isoflavones. All compounds (1-8) inhibited PTP1B in a dose dependent manner with IC50s ranging between 2.4 and 29.4μM. The most potent compound emerged to be prenylated isoflavone 5 (IC50=2.4μM). In kinetic studies, chromenedione derivatives (1-3) emerged to be reversible, competitive inhibitors, whereas prenylated isoflavones (5-8) were noncompetitive inhibitors.

Genistein and daidzein are predominant phytoestrogens for an estrogen alternative due to their higher receptor binding affinity. The present study illustrated the effective method to produce isoflavone riched soybean leaves (IRSL). Notably, ethylene application to soybean leaves under dark condition led a distinctive increase in genistin, daidzin, malonylgenistin and malonyldaidzin, which raised to total 25,911 μg/g of isoflavones in comparison to control leaves (>200 μg/g). Genistin and daidzin derivatives were enhanced with 120-fold (123→14,996 μg/g) and 185-fold (59→10,916 μg/g), respectively. The changes in metabolites by ethylene were systematically identified by UPLC–ESI-TOF/MS to give successfully PLS-DA score and heatmap analysis. The identified nine isoflavones were markedly increased, whereas rest main metabolites, kaempferols and soyasaponins were not affected. Furthermore, in contrast to the control leaves, the IRSL extract showed stimulation of collagen synthesis and induced gene expressions, in particular COL1A1 and COL3A1, in human dermal fibroblast.

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).

Human neutrophil elastase (HNE) represents a goodtherapeutic target for the treatment of inflammatory diseases aswell as invasion of microorganism. The methanol extract of aaerial part of Chelidonium majus L. showed high activity againstthe neutrophil elastase with an IC 50 value of 100µg/mL. Due toits potency, subsequent bioactivity-guided fractionation ofmethanol extract led to six alkaloids (1-6), which were identifiedas dihydrosanguinarine (1), (s)-stylopine (2), arnottianamide (3),(+)-chelidonine (4), spallidamine (5), and N-trans-feruloyltyramine (6). Among of them, three alkaloids (2, 5, and 6)inhibited HNE in a dose-dependent manner with IC 50 rangingbetween 11.6 and 51.0µM. Lineweaver-Burk and Dixon plots,and their secondary replots showed that alkaloids (2, 5, and 6)were mixed inhibitors of HNE. The analysis of K I and K IS valueproved that all inhibitors (2, 5, and 6) had reversible mixed typeI mechanism.Keywords Chelidonium majus L. · human neutrophil elastase ·inflammation · isoquinoline alkaloidIntroductionThe regulation of the enzymatic activity of human neutrophilelastase (HNE) has been the attractive field because neutrophilsare the first cells recruited to inflammatory sites and from theearliest line of defense against the invasion of microorganism(Brice et al., 2010). The human neutrophil elastase (EC 3. 4. 21.37) is the family of serine proteases that possess the ability tohydrolyze the extracellular matrix protein (Siedle et al., 2007). Itis present in azurophil granules in the neutrophil cytoplasm. HNEis a proteolytic enzyme involved in pathogenesis of emphysema,adult respiratory distress syndrome and rheumatoid arthritis(Crocetti et al., 2013). The catalytic site of HNE molecule iscomposed of the triad His41-Asp99-Ser173, of which the oxygenof serine attacks carbonyl group on the target substrate (Bode etal., 1989). The activity of HNE is controlled by inhibitor namedα1-antitrypsin produced in the liver. However, their HNE affinityis strongly decreased by oxidative stress and by proteases releasedfrom leukocytes that are recruited to inflammation sites (Brice etal., 2010). Thus, the imbalance between HNE and its inhibitors isimplicated in many inflammation diseases as like pulmonaryemphysema.Chelidonium majus L. is a perennial herbaceous plant of thefamily Papaveraceae, which is widely distributed around theworld. This plant has been traditionally used as an herbal medicinefor treatment of gastric ulcer, oral infection and liver disease(Lenfield et al., 1981). These effects are mainly due to the alkaloidspresent in milk sap. The major bioactive components of this plantare isoquinoline alkaloids such as chelidonine, chelerythrine,sanguianarine, bebeerine and coptisine (Barreto et al., 2003). Itincluded flavonoids and various acids such as ferulic, cumaric,caffeic and chelidonic acids (Barens J et al., 2007). The constituentsof C. majus L. have been found to exhibit antitumor, antiviral, andanti-inflammatory activities (Colombo and Bosisio, 1996). Additionally,the potent and selective acetylcholinesterase inhibition wasobserved on 8-hydroxydihydrochelerythrine in C. majus L (Cho etal., 2006).During a screening procedure on higher plant to find neutrophilelastase inhibitors, methanol extract of C. majus L. was shown toexhibit considerable inhibitory activity. In this study, we isolatedsix alkaloids targeting to HNE from the methanol extract of aerial

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.

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 β-glucosidase activity increased with 0.2 → 0.7%, 7.5 → 9.8 log cfu/mL, and 0.0 3 → 1.75 U/g in SPYs. Total average isoflavones were considerably reduced (3180.3 → 2018.3 μg/g) with the increase of aglycone contents (191.8 → 770.2 μg/g), especially, daidzein exhibited the most remarkable increase rate (98.6 → 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 → 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. Keywords: Fermented soybean, Isoflavone, CLA, Lactobacillus plantarum, Antioxidant

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.

Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).

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 β-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.

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-β- D -glucopyranoside (3), emodin-8-O-β- D -glucopyranoside (4), emodin-1-O-β- 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=5.4 μM, whereas its glucosides (4 and 5) had IC50=0.85 μM and 0.43 μM respectively. A similar trend was observed with physcion (1, IC50>200 μM) and its glucoside (3, IC50=6.2 μM). 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.

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson's disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC50 of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC50=500 nM), significantly. Another potent inhibitor 1 (IC50=2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: Ki(app)=1.48 nM, k3=0.0033 nM(-1) min(-1) and k4=0.0049 min(-1). Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC50. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.

Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).

Angelica keiskei is used as popular functional food stuff. However, quantitative analysis of this plant's metabolites has not yet been disclosed. The principal phenolic compounds (1-16) within A. keiskei were isolated, enabling us to quantify the metabolites within different parts of the plant. The specific quantification of metabolites (1-16) was accomplished by multiple reaction monitoring (MRM) using a quadruple tandem mass spectrometer. The limit of detection and limit of quantitation were calculated as 0.4-44 μg/kg and 1.5-148 μg/kg, respectively. Abundance and composition of these metabolites varied significantly across different parts of plant. For example, the abundance of chalcones (12-16) decreased as follows: root bark (10.51 mg/g)>stems (8.52 mg/g)>leaves (2.63 mg/g)>root cores (1.44 mg/g). The chalcones were found to be responsible for the xanthine oxidase (XO) inhibition shown by this plant. The most potent inhibitor, xanthoangelol inhibited XO with an IC50 of 8.5 μM. Chalcones (12-16) exhibited mixed-type inhibition characteristics.

Bacterial neuraminidase (NA) is a lynch pin enzyme in the formation of biofilms. Thus NA continues to be one of the key enzymes targeted by bacterial infection. The purpose of this manuscript is to communicate four new naturally derived inhibitors of neuraminidase (IC50s 3.7-24.4μM). All these active species (1-4) contained a resveratrol chemotype, however resveratrol itself was inactive (IC50>100μM). 1-4 were isolated from the 60% aqueous ethanol extract of seeds of Paeonia lactiflora, which exhibited potent neuraminidase inhibition. Purification of the extracts yielded four chiral polyphenols, suffruticosol A (1), suffruticosol B (2), trans-e-viniferin (3), and trans-gnetin H (4). Mechanistic analysis of 1-4's inhibition showed that they were all reversible, noncompetitive inhibitors. Trans-e-viniferin (3) underwent trans-cis isomerization, which led to a reduction in inhibition potency. This correlates with the fact that the cis-isomer is a weaker inhibitor of neuraminidase than the trans-isomer. Importantly, significantly different optical rotations ([α]D) compared to previous reports were found for suffruticosols A (+95 vs -34) and B (+136 vs +13). These two species are the most important standard metabolites in the whole paeoniaceae family and therefore correction of this error is important.