Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera.

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

Induction of ER Stress-Mediated Apoptosis by the Major Component 5,7,4'-Trimethoxyflavone Isolated from Kaempferia parviflora Tea Infusion.

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

Seasonal Variation and Possible Biosynthetic Pathway of Ginsenosides in Korean Ginseng Panax ginseng Meyer.

Whereas Korean ginseng, Panax ginseng Meyer, is harvested in the fall, the variation of ginsenoside content in field-grown ginseng across seasonal development has never been investigated in Korea. Thus, ultra-high performance liquid chromatography (UHPLC) analysis of nine major ginsenosides, including ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rd, and Ro, in the roots of five-year-old P. ginseng cultivated in Bongwha, Korea in 2017 was performed. The total ginsenoside content changed as many as three times throughout the year, ranging from 1.37 ± 0.02 (dry wt %) in January to 4.26 ± 0.03% in May. Total ginsenoside content in the harvest season was 2.49 ± 0.03%. Seasonal variations of panaxadiol-type ginsenosides (PPD) and panaxatriol-type ginsenosides (PPT) were found to be similar, but more PPD was always measured. However, the seasonal variation of oleanolic acid-type ginsenoside, Ro, was different from that of PPD and PPT, and the highest Ro content was observed in May. The ratio of PPD/PPT, as well as other representative ginsenosides, was compared throughout the year. Moreover, the percent composition of certain ginsenosides in both PPD and PPT types was found to be in a complementary relationship each other, which possibly reflected the biosynthetic pathway of the related ginsenosides. This finding would not only provide scientific support for the production and quality control of the value-added ginseng products, but also facilitate the elucidation of the ginsenoside biosynthetic pathway.

Icariin Metabolism by Human Intestinal Microflora.

Icariin is a major bioactive compound of Epimedii Herba, a traditional oriental medicine exhibiting anti-cancer, anti-inflammatory and anti-osteoporosis activities. Recently, the estrogenic activities of icariin drew significant attention, but the published scientific data seemed not to be so consistent. To provide fundamental information for the study of the icaritin metabolism, the biotransformation of icariin by the human intestinal bacteria is reported for the first time. Together with human intestinal microflora, the three bacteria Streptococcus sp. MRG-ICA-B, Enterococcus sp. MRG-ICA-E, and Blautia sp. MRG-PMF-1 isolated from human intestine were reacted with icariin under anaerobic conditions. The metabolites including icariside II, icaritin, and desmethylicaritin, but not icariside I, were produced. The MRG-ICA-B and E strains hydrolyzed only the glucose moiety of icariin, and icariside II was the only metabolite. However, the MRG-PMF-1 strain metabolized icariin further to desmethylicaritin via icariside II and icaritin. From the results, along with the icariin metabolism by human microflora, it was evident that most icariin is quickly transformed to icariside II before absorption in the human intestine. We propose the pharmacokinetics of icariin should focus on metabolites such as icariside II, icaritin and desmethylicaritin to explain the discrepancy between the in vitro bioassay and pharmacological effects.

Deglycosylation of isoflavone C-glycosides by newly isolated human intestinal bacteria.

BACKGROUND: Plant isoflavones are mostly present in the glycoside form. Isoflavone aglycones produced by intestinal microflora are reported to be more bioactive than the glycoside form. However, the deglycosylation of isoflavone C-glycosides is known to be rare, and is less studied. RESULTS: Three new bacteria were isolated from human faecal samples, two of which hydrolysed the C-glycosidic bond of puerarin, daidzein-8-C-glucoside. They were identified as two Lactococcus species, herein designated as MRG-IFC-1 and MRG-IFC-3, and an Enterococcus species, herein designated MRG-IFC-2, based on their 16S rDNA sequences. From a reactivity study, it was found that Lactococcus sp. MRG-IFC-1 and Enterococcus sp. MRG-IFC-2 hydrolysed isoflavone C- and O-glycosides, as well as the flavone O-glycoside apigetrin, but could not hydrolyse the flavone C-glycosidic bond of vitexin. The other Lactococcus sp., MRG-IF-3, could not hydrolyse the C-glycosidic linkage of puerarin, while it showed a broad substrate spectrum of O-glycosidase activity similar to the other two bacteria. Puerarin was completely converted to daidzein within 100 min by Lactococcus sp. MRG-IFC-1 and Enterococcus sp. MRG-IFC-2, which is the fastest conversion among the reported human intestinal bacteria. CONCLUSION: Two new puerarin-metabolising human intestinal bacteria were isolated and identified, and the deglycosylation activity for various flavonoid glycosides was investigated. The results could facilitate the study of C-glycosidase reaction mechanisms, as well as the pharmacokinetics of bioactive C-glycoside natural products.

Chiroptical study and absolute configuration of (-)-O-DMA produced from daidzein metabolism.

To elucidate the hitherto unknown absolute configuration of (-)-O-desmethylangolensin ((-)-O-DMA), an intestinal bacterial metabolite produced from daidzein, chiroptical study, including specific optical rotation and electronic circular dichroism (ECD), of (R)-O-DMA was carried out by Time-Dependent Density Functional Theory (TD-DFT) calculations. Intramolecular hydrogen bonding between 2'-OH and carbonyl oxygen at 1-C of O-DMA was a governing factor for O-DMA to form the stable conformations. Total energy values of four possible conformers were calculated in the framework of DFT using the B3LYP exchange correlation functional at the 6-31++G basis set level. The theoretical specific rotation and ECD spectra of all conformers in ethanol were obtained by TD-DFT calculation using B3LYP functional at the 6-311++G basis set level, and compared to the experimental data. Chiroptical properties of (R)-O-DMA showed a good agreement with the biological (-)-O-DMA. Therefore, the stereospecific biosynthetic pathway of (-)-O-DMA was proposed as daidzein → (R)-dihydrodaidzein ↔ (S)-dihydrodaizein → (R)-O-DMA.

Flavonoids Biotransformation by Human Gut Bacterium Dorea sp. MRG-IFC3 Cell-Free Extract.

Human gut bacterium Dorea sp. MRG-IFC3 is unique in that it is capable of metabolizing puerarin, an isoflavone C-glycoside, whereas it shows broad substrate glycosidase activity for the various flavonoid O-glycosides. To address the question on the substrate specificity, as well as biochemical characteristics, cell-free biotransformation of flavonoid glycosides was performed under various conditions. The results showed that there are two different enzyme systems responsible for the metabolism of flavonoid C-glycosides and O-glycosides in the MRG-IFC3 strain. The system responsible for the conversion of puerarin was inducible and comprised of two enzymes. One enzyme oxidizes puerarin to 3"-oxo-puerarin and the other enzyme converts 3"-oxo-puearin to daidzein. The second enzyme was only active toward 3"-oxo-puerarin. The activity of puerarin conversion to daidzein was enhanced in the presence of Mn2+ and NAD+. It was concluded that the puerarin C-deglycosylation by Dorea sp. MRG-IFC3 possibly adopts the same biochemical mechanism as the strain PUE, a species of Dorea longicatena.

Amino acid substitutions in naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 result in regio- and stereo-specific hydroxylation of flavanone and isoflavanone.

Wild-type naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 transforms relatively planar flavone and isoflavone to cis-dihydrodiols. However, this enzyme cannot catalyze the transformation of flavanone and isoflavanone in which a phenyl group bonds to the stereogenic C2 or C3 of the C-ring. Protein modeling suggested that Phe224 in the substrate binding site of NDO may play a key role in substrate specificity toward flavanone and isoflavanone. Site-directed mutants of NDO with substitution of Phe224 with Tyr biotransformed only the (S)-stereoisomers of flavanone and isoflavanone, producing an 8-OH group on the A-ring. In contrast, the Phe224Cys and Phe224Gln substitutions, which used (2S)-flavanone as a substrate, and Phe224Lys, which transformed (2S)-flavanone and (3S)-isoflavanone, each showed lower activity than the Phe224Tyr substitution. The remainder of the tested mutants had no activity with flavanone and isoflavanone. Protein docking studies of flavanone and isoflavanone to the modeled mutant enzyme structures revealed that an expanded substrate binding site, due to mutation at 224, as well as appropriate hydrophobic interaction with the residue at 224, are critical for successful binding of the substrates. Results of this study also suggested that in addition to the previously known Phe352, the Phe224 site of NDO appears to be important site for expanding the substrate range of NDO and bringing regiospecific and stereospecific hydroxylation reactions to C8 of the flavanone and isoflavanone A-rings.

Theoretical study on the glycosidic C-C bond cleavage of 3''-oxo-puerarin.

Puerarin, daidzein C-glucoside, was known to be biotransformed to daidzein by human intestinal bacteria, which is eventually converted to (S)-equol. The metabolic pathway of puerarin to daidzein by DgpABC of Dorea sp. PUE strain was reported as puerarin (1) → 3''-oxo-puerarin (2) → daidzein (3) + hexose enediolone (C). The second reaction is the cleavage of the glycosidic C-C bond, supposedly through the quinoid intermediate (4). In this work, the glycosidic C-C bond cleavage reaction of 3''-oxo-puerarin (2) was theoretically studied by means of DFT calculation to elucidate chemical reaction mechanism, along with biochemical energetics of puerarin metabolism. It was found that bioenergetics of puerarin metabolism is slightly endergonic by 4.99 kcal/mol, mainly due to the reaction step of hexose enediolone (C) to 3''-oxo-glucose (A). The result implied that there could be additional biochemical reactions for the metabolism of hexose enediolone (C) to overcome the thermodynamic energy barrier of 4.59 kcal/mol. The computational study focused on the C-C bond cleavage of 3''-oxo-puerarin (2) found that formation of the quinoid intermediate (4) was not accessible thermodynamically, rather the reaction was initiated by the deprotonation of 2''C-H proton of 3''-oxo-puerarin (2). The 2''C-dehydro-3''-oxo-puerarin (2a2C) anionic species produced hexose enediolone (C) and 8-dehydro-daidzein anion (3a8), and the latter quickly converted to daidzein through the daidzein anion (3a7). Our study also explains why the reverse reaction of C-glycoside formation from daidzein (3) and hexose enediolone (C) is not feasible.

C-Glycoside-Metabolizing Human Gut Bacterium, Dorea sp. MRG-IFC3.

Biochemical gut metabolism of dietary bioactive compounds is of great significance in elucidating health-related issues at the molecular level. In this study, a human gut bacterium cleaving C-C glycosidic bond was screened from puerarin conversion to daidzein, and a new, gram-positive C-glycoside-deglycosylating strain, Dorea sp. MRG-IFC3, was isolated from human fecal sample under anaerobic conditions. Though MRG-IFC3 biotransformed isoflavone C-glycoside, it could not metabolize other C-glycosides, such as vitexin, bergenin, and aloin. As evident from the production of the corresponding aglycons from various 7-O-glucosides, MRG-IFC3 strain also showed 7-O-glycoside cleavage activity; however, flavone 3-O-glucoside icariside II was not metabolized. In addition, for mechanism study, C-glycosyl bond cleavage of puerarin by MRG-IFC3 strain was performed in D2O GAM medium. The complete deuterium enrichment on C-8 position of daidzein was confirmed by 1H NMR spectroscopy, and the result clearly proved for the first time that daidzein is produced from puerarin. Two possible reaction intermediates, the quinoids and 8-dehydrodaidzein anion, were proposed for the production of daidzein-8d. These results will provide the basis for the mechanism study of stable C-glycosidic bond cleavage at the molecular level.

Evaluation of Chemical Compositions and the Antioxidant and Cytotoxic Properties of the Aqueous Extract of Tri-Yannarose Recipe (Areca catechu, Azadirachta indica, and Tinospora crispa).

Tri-Yannarose is a Thai traditional herbal medicine formula composed of Areca catechu, Azadirachta indica, and Tinospora crispa. It possesses antipyretic, diuretic, expectorant, and appetite-stimulating effects. This study aimed to evaluate the antioxidant activities, cytotoxicity, and chemical constituents of an aqueous extract following a Tri-Yannarose recipe and its plant ingredients. The phytochemical analysis was performed using LC-QTOF-MS. Antioxidant activities were determined using DPPH, ABTS, TPC, TFC, FRAP, NBT, MCA, and ORAC assays. Cytotoxicity was investigated using a methyl thiazol tetrazolium (MTT) assay. In addition, the relationship between the chemical composition of Tri-Yannarose and antioxidant activities was investigated by examining the structure-activity relationship (SAR). The results of the LC-QTOF-MS analysis revealed trigonelline, succinic acid, citric acid, and other chemical constituents. The aqueous extract of the recipe showed significant scavenging effects against ABTS and DPPH radicals, with IC50 values of 1054.843 ± 151.330 and 747.210 ± 44.173 µg/mL, respectively. The TPC of the recipe was 92.685 mg of gallic acid equivalent/g of extract and the TFC was 14.160 mg of catechin equivalent/g of extract. All extracts demonstrated lower toxicity in the Vero cell line according to the MTT assay. In addition, the SAR analysis indicated that prenyl arabinosyl-(1-6)-glucoside and quinic acid were the primary antioxidant compounds in the Tri-Yannarose extract. In conclusion, this study demonstrates that Tri-Yannarose and its plant ingredients have potent antioxidant activities with low toxicity. These results support the application of the Tri-Yannarose recipe for the management of a range of disorders related to oxidative stress.

Research Progress on Natural Diterpenoids in Reversing Multidrug Resistance.

Multidrug resistance (MDR) is one of the main impediments in successful chemotherapy in cancer treatment. Overexpression of ATP-binding cassette (ABC) transporter proteins is one of the most important mechanisms of MDR. Natural products have their unique advantages in reversing MDR, among which diterpenoids have attracted great attention of the researchers around the world. This review article summarizes and discusses the research progress on diterpenoids in reversing MDR.

Allyl Aryl Ether Cleavage by Blautia sp. MRG-PMF1 Cocorrinoid O-Demethylase.

Coabalamin-dependent O-demethylase in Blautia sp. strain MRG-PMF1 was found to catalyze the unprecedented allyl aryl ether cleavage reaction. To expand the potential biotechnological applications, the reaction mechanism of the allyl aryl ether C-O bond cleavage, proposed to utilize the reactive Co(I) supernucleophile species, was studied further from the anaerobic whole-cell biotransformation. Various allyl naphthyl ether derivatives were reacted with Blautia sp. MRG-PMF1 O-demethylase, and stereoisomers of allyl naphthyl ethers, including prenyl and but-2-enyl naphthyl ethers, were converted to the corresponding naphthol in a stereoselective manner. The allyl aryl ether cleavage reaction was regioselective, and 2-naphthyl ethers were converted faster than the corresponding 1-naphthyl ethers. However, MRG-PMF1 cocorrinoid O-demethylase was not able to convert (2-methylallyl) naphthyl ether substrates, and the conversion of propargyl naphthyl ether was extremely slow. From the results, it was proposed that the allyl ether cleavage reaction follows the nucleophilic conjugate substitution (SN2') mechanism. The reactivity and mechanism of the new allyl ether cleavage reaction by cobalamin-dependent O-demethylase would facilitate the application of Blautia sp. MRG-PMF1 O-demethylase in the area of green biotechnology. IMPORTANCE Biodegradation of environmental pollutants and valorization of biomaterials in a greener way is of great interest. Cobalamin-dependent O-demethylase in Blautia sp. MRG-PMF1 exclusively involves anaerobic C1 metabolism by cleaving the C-O bond of aromatic methoxy group and also produces various aryl alcohols by metabolizing allyl aryl ether compounds. Whereas methyl ether cleavage reaction is known to follow the SN2' mechanism, the reaction pattern and mechanism of the new allyl ether cleavage reaction by cobalamin-dependent O-demethylase have never been studied. For the first time, stereoselectivity and the SN2' mechanism of allyl aryl ether cleavage reaction by Blautia sp. MRG-PMF1 O-demethylase is reported, and the results would facilitate the application of Blautia sp. MRG-PMF1 O-demethylase in the area of green biotechnology.

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Stereospecific microbial production of isoflavanones from isoflavones and isoflavone glucosides.

A Gram-negative anaerobic microorganism, MRG-1, isolated from human intestine showed high activities of deglycosylation and reduction of daidzin, based on rapid TLC analysis. A rod-shaped strain MRG-1 was identified as a new species showing 91.0% homology to Coprobacillus species, based on 16S rRNA sequence analysis. The strain MRG-1 showed ß-glucosidase activity toward daidzin and genistin, and daidzein and genistein were produced, respectively. However, the strain MRG-1 did not react with flavone glycosides, flavanone glycosides, and isoflavone C-glucoside. Besides, MRG-1 showed stereoselective reductase activity to isoflavone, daidzein, genistein, 7-hydroxyisoflavone, and formononetin, resulting in the formation of corresponding R-isoflavanone enantiomers. The new isoflavanones of 7-hydroxyisoflavanone and dihydroformononetin were characterized by NMR, and the absolute configurations of the enantiomers were determined with CD spectroscopy. The kinetic study of the anaerobic biotransformation showed both activities were exceptionally fast compared to the reported conversion by other anaerobic bacteria.

Flavonoids biotransformation by bacterial non-heme dioxygenases, biphenyl and naphthalene dioxygenase.

This review details recent progresses in the flavonoid biotransformation by bacterial non-heme dioxygenases, biphenyl dioxygenase (BDO), and naphthalene dioxygenase (NDO), which can initially activate biphenyl and naphthalene with insertion of dioxygen in stereospecfic and regiospecific manners. Flavone, isoflavone, flavanone, and isoflavanol were biotransformed by BDO from Pseudomonas pseudoalcaligenes KF707 and NDO from Pseudomonas sp. strain NCIB9816-4, respectively. In general, BDO showed wide range of substrate spectrum and produced the oxidized products, whereas NDO only metabolized flat two-dimensional substrates of flavone and isoflavone. Furthermore, biotransformation of B-ring skewed substrates, flavanone and isoflavanol, by BDO produced the epoxide products, instead of dihydrodiols. These results support the idea that substrate-driven reactivity alteration of the Fe-oxo active species may occur in the active site of non-heme dioxygenases. The study of flavonoid biotransformation by structurally-well defined BDO and NDO will provide the substrate structure and reactivity relationships and eventually establish the production of non-plant-originated flavonoids by means of microbial biotechnology.

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707.

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

Laparoscopy-assisted distal gastrectomy compared to open distal gastrectomy in early gastric cancer.

BACKGROUND: This study aimed to evaluate the feasibility of laparoscopy-assisted distal gastrectomy (LADG) in early gastric cancer (EGC) with special interest in a learning curve effect. METHODS: The clinical outcomes of EGC patients who underwent LADG (n = 100) and sex-, age- and body mass index- (BMI) matched EGC patients who underwent open distal gastrectomy (ODG; n = 100) were compared retrospectively. In addition, the outcomes between the early (n = 50) and late LADG group (n = 50) were compared. RESULTS: The mean number of retrieved lymph nodes was significantly smaller in the LADG group than in the ODG group (29.3 vs. 36.4, p < 0.001). The operative time of the LADG group was significantly longer than in the ODG group (249.1 vs. 152.9 min, p < 0.001). The complication rates were comparable between both groups (14 vs. 13%, p = 0.84). No cancer-related death was observed in either group. Between early and late LADG groups, the operative time was shorter (p < 0.001) and the number of retrieved lymph nodes was higher (p = 0.016) in the late group. CONCLUSIONS: LADG seems to be a safe and feasible procedure in treating EGC, as it shows comparable outcomes with ODG. The potential disadvantages of LADG, such as longer operation time and smaller number of retrieved lymph nodes, diminished after overcoming the learning curve.

Conversion of (3S,4R)-tetrahydrodaidzein to (3S)-equol by THD reductase: proposed mechanism involving a radical intermediate.

To elucidate the mechanism of (3S)-equol biosynthesis, (2,3,4-d(3))-trans-THD was synthesized and converted to (3S)-equol by THD reductase in Eggerthella strain Julong 732. The position of the deuterium atoms in (3S)-equol was determined by (1)H NMR and (2)H NMR spectroscopy, and the product was identified as (2,3,4(alpha)-d(3))-(3S)-equol. All the deuterium atoms were retained, while the OH group at C-4 was replaced by a hydrogen atom with retention of configuration. To explain the deuterium retention in this stereospecific reduction, we propose a mechanism involving radical intermediates.

Time-dependent density functional theory-assisted absolute configuration determination of cis-dihydrodiol metabolite produced from isoflavone by biphenyl dioxygenase.

Escherichia coli cells containing the biphenyl dioxygenase genes bphA1A2A3A4 from Pseudomonas pseudoalcaligenes KF707 were found to biotransform isoflavone and produced a metabolite that was not found in a control experiment. Liquid chromatography/mass spectrometry (LC/MS) and (1)H and (13)C nuclear magnetic resonance (NMR) analyses indicated that biphenyl dioxygenase induced 2',3'-cis-dihydroxylation of the B-ring of isoflavone. In a previous report, the same enzyme showed dioxygenase activity toward flavone, producing flavone 2',3'-cis-dihydrodiol. Due to growing interest in flavone chemistry and the absolute configuration of natural products, time-dependent density functional theory (TD-DFT) calculations were combined with circular dichroism (CD) spectroscopy to determine the absolute configuration of the isoflavone dihydrodiol. By computational methods, the structure of the isoflavone metabolite was determined to be 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. This structure was confirmed further by the modified Mosher's method. The same protocol was applied to the flavone metabolite, and the absolute configuration was determined to be 2-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. After determination of the absolute configurations of the biotransformation products, we suggest the binding mode of these substrate analogs to the enzyme active site.