Inhibitory Potential of Quercetin Derivatives Isolated from the Aerial Parts of Siegesbeckia pubescens Makino against Bacterial Neuraminidase.

This study aimed to isolate bacterial neuraminidase (BNA) inhibitory O-methylated quercetin derivatives from the aerial parts of S. pubescens. All the isolated compounds were identified as O-methylated quercetin (1-4), which were exhibited to be noncompetitive inhibitors against BNA, with IC50 ranging from 14.0 to 84.1 µM. The responsible compounds (1-4) showed a significant correlation between BNA inhibitory effects and the number of O-methyl groups on quercetin; mono (1, IC50 = 14.0 µM) > di (2 and 3, IC50 = 24.3 and 25.8 µM) > tri (4, IC50 = 84.1 µM). In addition, the binding affinities between BNA and inhibitors (1-4) were also examined by fluorescence quenching effect with the related constants (KSV, KA, and n). The most active inhibitor 1 possessed a KSV with 0.0252 × 105 L mol-1. Furthermore, the relative distribution of BNA inhibitory O-methylated quercetins (1-4) in S. pubescens extract was evaluated using LC-Q-TOF/MS analysis.

Amorphigenin from Amorpha fruticosa L. Root Extract Induces Autophagy-Mediated Melanosome Degradation in mTOR-Independent- and AMPK-Dependent Manner.

In this study, we investigated the depigmentation effect of Amorpha fruticosa L. root extract (RE), an herbal medicine. A. fruticosa RE significantly induced depigmentation in α-MSH-treated B16F10 cells at noncytotoxic concentrations. Further, the RE decreased the protein levels of the melanosomal proteins Tyr and Pmel without decreasing their transcript levels. We found that MG132, a proteasome complex inhibitor, was unable to rescue the protein levels, but PepA/E-64D (a lysosomal enzyme inhibitor), 3-MA (a representative autophagy inhibitor), and ATG5 knockdown effectively rescued the protein levels and inhibited the depigmentation effect following RE treatment. Among rotenoids, amorphigenin composed in the RE was identified as a functional chemical that could induce depigmentation; whereas rapamycin, an mTOR inhibitor and a nonselective autophagy inducer, could not induce depigmentation, and amorphigenin effectively induced depigmentation through the degradation of melanosomal proteins. Amorphigenin activated AMPK without affecting mTOR, and knockdown of AMPK offset the whitening effect through degradation of melanosome proteins by amorphigenin. Results from this study suggested that amorphigenin can induce degradation of the melanosome through an AMPK-dependent autophagy process, and has the potential to be used as a depigmentation agent for the treatment of hyperpigmentation.

New dihydrobenzoxanthone derivatives with bacterial neuraminidase inhibitory activity isolated from Artocarpus elasticus.

Artocarpus elasticus is a popular fruit tree in the tropical regions. Primary screenings of methanol extracts of the root bark confirmed its potent inhibition of bacterial neuraminidase (BNA), which plays an essential role in the pathogenesis of many microbial diseases. Assessments of the responsible phytochemicals were conducted by isolating eight compounds (1-8) and two of them (6 and 8) were identified as new compounds. Among the isolates, the dihydrobenzoxanthones attained the highest BNA inhibition with IC50 values of 0.5 âˆ¼ 3.9 µM. Further investigation of the inhibitory mechanism by Lineweaver-Burk plots revealed the phytochemicals to function as reversible noncompetitive inhibitors. Fluorescence quenching showed their binding affinities were highly correlated with their inhibitory potential dose-dependently. Molecular docking experiments suggested the dihydrobenzoxanthones (4 and 6) as noncompetitive inhibitors of BNA with unique interaction with Tyr435 of BNA in comparison with the mother flavonoid (7).

Iridal-Type Triterpenoids Displaying Human Neutrophil Elastase Inhibition and Anti-Inflammatory Effects from Belamcanda chinensis.

The aim of this study is to explore anti-inflammatory phytochemicals from B. chinensis based on the inhibition of pro-inflammatory enzyme, human neutrophil elastase (HNE) and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage. Three stereoisomers of iridal-type triterpenoids (1-3) were isolated from the roots of B. chinensis and their stereochemistries were completely identified by NOESY spectra. These compounds were confirmed as reversible noncompetitive inhibitors against HNE with IC50 values of 6.8-27.0 µM. The binding affinity experiment proved that iridal-type triterpenoids had only a single binding site to the HNE enzyme. Among them, isoiridogermanal (1) and iridobelamal A (2) displayed significant anti-inflammatory effects by suppressing the expressions of pro-inflammatory cytokines, such as iNOS, IL-1ß, and TNF-α through the NF-κB pathway in LPS-stimulated RAW264.7 cells. This is the first report that iridal-type triterpenoids are considered responsible phytochemicals for anti-inflammatory effects of B. chinensis.

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.

Xanthine Oxidase Inhibition and Anti-LDL Oxidation by Prenylated Isoflavones from Flemingia philippinensis Root.

Xanthine oxidase is a frontier enzyme to produce oxidants, which leads to inflammation in the blood. Prenylated isoflavones from Flemingia philippinensis were found to display potent inhibition against xanthine oxidase (XO). All isolates (1-9) inhibited XO enzyme with IC50 ranging 7.8~36.4 µM. The most active isoflavones (2-5, IC50 = 7.8~14.8 µM) have the structural feature of a catechol motif in B-ring. Inhibitory behaviors were disclosed as a mixed type I mode of inhibition with KI < KIS. Binding affinities to XO enzyme were evaluated. Fluorescence quenching effects agreed with inhibitory potencies (IC50s). The compounds (2-5) also showed potent anti-LDL oxidation effects in the thiobarbituric acid-reactive substances (TBARS) assay, the lag time of conjugated diene formation, relative electrophoretic mobility (REM), and fragmentation of apoB-100 on copper-mediated LDL oxidation. The compound 4 protected LDL oxidation with 0.7 µM in TBARS assay, which was 40-fold more active than genistein (IC50 = 30.4 µM).

Tyrosinase Inhibition and Kinetic Details of Puerol A Having But-2-Enolide Structure from Amorpha fruticosa.

Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 µM) and diphenolase (IC50 = 3.8 µM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k3 = 0.0279 µM-1 min-1 and k4 = 0.003 min-1. A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with Emet. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 µM. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 µM.

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.

Tyrosinase inhibitory study of flavonolignans from the seeds of Silybum marianum (Milk thistle).

Anti-melanogenesis effects of silymarin from milk thistle have been reported recently, but detailed tyrosinase inhibition properties of individual components have not been investigated. This study purported to substantiate tyrosinase inhibition and its mechanism based on a single metabolite. The responsible components for tyrosinase inhibition of target source were found out as flavonolignans which consist of isosilybin A (1), isosilybin B (2), silydianin (3), 2,3-dihydrosilychristin (4), silychristin A (5), silychristin B (6) and silybin (7), respectively. The isolated flavonolignans (1-7) inhibited both monophenolase (IC50 = 1.7-7.6 µM) and diphenolase (IC50 = 12.1-44.9 µM) of tyrosinase significantly. Their inhibitions were 10-fold effective in comparison with their mother skeletons (8-10). Inhibitory functions were also proved by HPLC analysis using N-acetyl-l-tyrosine as substrate. The predominant formation of Emet·I was confirmed from a long prolongation of lag time and a decrease of the static state activity of the enzyme. All tested compounds had a significant binding affinity to tyrosinase with KSV values of 0.06-0.27 × 104 L·mol-1, which are well correlated with IC50s. In kinetic study, all flavonolignan (1-7) were mixed type I (KI < KIS) inhibitors, whereas their mother skeletons (8-10) were competitive ones. The UPLC-ESI-TOF/MS analysis showed that the isolated inhibitors are the most abundant metabolites in the target plant.

Human neutrophil elastase (HNE) inhibitory polyprenylated acylphloroglucinols from the flowers of Hypericum ascyron.

In the course of an investigation of human neutrophil elastase (HNE) associated with inflammation, the extract of the flower parts of Hypericum ascyron showed a significant influence to HNE. The responsible metabolites to HNE inhibition were found to be eight polyprenylated acylphloroglucinols, PPAPs (1-8) which showed IC50 ranges between 2.4 and 19.9 µM. This is the first report to demonstrate that PPAP skeleton exhibits potent HNE inhibition. The compounds 1-3 were characterized and newly named as ascyronone E (IC50 = 4.3 µM), ascyronone F (IC50 = 19.9 µM), ascyronone G (IC50 = 4.5 µM) based on 2D-NMR spectroscopic data. In the kinetic analysis of double reciprocal plots, all the compounds showed noncompetitive behaviors to HNE enzyme with the remaining of Km and the increase of Vmax. The binding affinity levels (KSV) by using fluorescence were sufficient to be able to prove that PPAPs (1-8) had compliant interaction with inhibitory potencies.

Competitive α-glucosidase inhibitors, dihydrobenzoxanthones, from the barks of Artocarpus elasticus.

This study aimed to search the α-glucosidase inhibitors from the barks part of Artocarpus elasticus. The responsible compounds for α-glucosidase inhibition were found out as dihydrobenzoxanthones (1-4) and alkylated flavones (5-6). All compounds showed a significant enzyme inhibition toward α-glucosidase with IC50s of 7.6-25.4 µM. Dihydrobenzoxanthones (1-4) exhibited a competitive inhibition to α-glucosidase. This competitive behaviour was fully characterised by double reciprocal plots, Yang's method, and time-dependent experiments. The compound 1 manifested as the competitive and reversible simple slow-binding, with kinetic parameters k3 = 0.0437 µM-1 min-1, k4 = 0.0166 min-1, and Kiapp = 0.3795 µM. Alkylated flavones (5-6) were mixed type I (KI < KIS) inhibitors. The binding affinities (KSV) represented by all inhibitors were correlated to their concentrations and inhibitory potencies (IC50). Moreover, compounds 1 and 5 were identified as new ones named as artoindonesianin W and artoflavone B, respectively. Molecular modelling study proposed the putative binding conformation of competitive inhibitors (1-4) to α-glucosidase at the atomic level.

Effectiveness of Prenyl Group on Flavonoids from Epimedium koreanum Nakai on Bacterial Neuraminidase Inhibition.

In this study, the inhibitory potential of bacterial neuraminidase (NA) was observed on the leaves of Epimedium koreanum Nakai, which is a popular ingredient in traditional herbal medicine. This study attempted to isolate the relevant, responsible metabolites and elucidate their inhibition mechanism. The methanol extraction process yielded eight flavonoids (1⁻8), of which compounds 7 and 8 were new compounds named koreanoside F and koreanoside G, respectively. All the compounds (1⁻8) showed a significant inhibition to bacterial NA with IC50 values of 0.17⁻106.3 µM. In particular, the prenyl group on the flavonoids played a critical role in bacterial NA inhibition. Epimedokoreanin B (compound 1, IC50 = 0.17 µM) with two prenyl groups on C8 and C5' of luteolin was 500 times more effective than luteolin (IC50 = 85.6 µM). A similar trend was observed on compound 2 (IC50 = 0.68 µM) versus dihydrokaempferol (IC50 = 500.4 µM) and compound 3 (IC50 = 12.6 µM) versus apigenin (IC50 = 107.5 µM). Kinetic parameters (Km, Vmax, and Kik/Kiv) evaluated that all the compounds apart from compound 5 showed noncompetitive inhibition. Compound 5 was proven to be a mixed type inhibitor. In an enzyme binding affinity experiment using fluorescence, affinity constants (KSV) were tightly related to inhibitory activities.

A Multi-Omics Analysis of Glycine max Leaves Reveals Alteration in Flavonoid and Isoflavonoid Metabolism Upon Ethylene and Abscisic Acid Treatment.

Phytohormones are central to plant growth and development. Despite the advancement in our knowledge of hormone signaling, downstream targets, and their interactions upon hormones action remain largely fragmented, especially at the protein and metabolite levels. With an aim to get new insight into the effects of two hormones, ethylene (ET) and abscisic acid (ABA), this study utilizes an integrated proteomics and metabolomics approach to investigate their individual and combined (ABA+ET) signaling in soybean leaves. Targeting low-abundance proteins, our previously established protamine sulfate precipitation method was applied, followed by label-free quantification of identified proteins. A total of 4129 unique protein groups including 1083 differentially modulated in one (individual) or other (combined) treatments were discerned. Functional annotation of the identified proteins showed an increased abundance of proteins related to the flavonoid and isoflavonoid biosynthesis and MAPK signaling pathway in response to ET treatment. HPLC analysis showed an accumulation of isoflavones (genistin, daidzein, and genistein) upon ET treatment, in agreement with the proteomics results. A metabolome analysis assigned 79 metabolites and further confirmed the accumulation of flavonoids and isoflavonoids in response to ET. A potential cross-talk between ET and MAPK signaling, leading to the accumulation of flavonoids and isoflavonoids in soybean leaves is suggested.

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.

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.

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).

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.

Crystal structure of the catalytic domain of Clostridium perfringens neuraminidase in complex with a non-carbohydrate-based inhibitor, 2-(cyclohexylamino)ethanesulfonic acid.

Anti-bacterial and anti-viral neuraminidase agents inhibit neuraminidase activity catalyzing the hydrolysis of terminal N-acetylneuraminic acid (Neu5Ac) from glycoconjugates and help to prevent the host pathogenesis that lead to fatal infectious diseases including influenza, bacteremia, sepsis, and cholera. Emerging antibiotic and drug resistances to commonly used anti-neuraminidase agents such as oseltamivir (Tamiflu) and zanamivir (Relenza) have highlighted the need to develop new anti-neuraminidase drugs. We obtained a serendipitous complex crystal of the catalytic domain of Clostridium perfringens neuraminidase (CpNanICD) with 2-(cyclohexylamino)ethanesulfonic acid (CHES) as a buffer. Here, we report the crystal structure of CpNanICD in complex with CHES at 1.24 Å resolution. Amphipathic CHES binds to the catalytic site of CpNanICD similar to the substrate (Neu5Ac) binding site. The 2-aminoethanesulfonic acid moiety and cyclohexyl groups of CHES interact with the cluster of three arginine residues and with the hydrophobic pocket of the CpNanICD catalytic site. In addition, a structural comparison with other bacterial and human neuraminidases suggests that CHES could serve as a scaffold for the development of new anti-neuraminidase agents targeting CpNanI.

Depigmentation of α-melanocyte-stimulating hormone-treated melanoma cells by ß-mangostin is mediated by selective autophagy.

Melanogenesis is a key pathway for the regulation of skin pigmentation and the development of skin-lightening/skin-whitening drugs or cosmetics. In this study, we found that ß-mangostin from seedcases of Garcinia mangostana inhibited α-melanocyte-stimulating hormone (α-MSH)-mediated melanogenesis in B16F10 melanoma cells and a three-dimensional human skin model. ß-Mangostin significantly inhibited the protein level of tyrosinase induced by α-MSH in UPS (ubiquitin proteasome system)-independent and lysosome-dependent manner. The inhibition of autophagy by 3-methyladenine treatment or ATG5 knockdown effectively recovered premelanosome protein as well as tyrosinase degraded by the ß-mangostin treatment. However, rapamycin, a representative non-selective autophagy inducer, triggered autophagy in α-MSH-stimulated cells, which was characterized by a considerable decrease in p62, but it was unable to inhibit melanogenesis. Melanosome-engulfing autophagosomes were observed using transmission electron microscopy. Furthermore, previously formed melanin could be degraded effectively in an autophagy-dependent manner in ß-mangostin-treated cells. Taken together, our results suggest that ß-mangostin inhibits the melanogenesis induced by α-MSH via an autophagy-dependent mechanism, and thus, the depigmentation effect of ß-mangostin may depend on autophagy targeted at the melanosome rather than non-selective autophagy.

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.