The lignan glycosyltransferase UGT236(belonging to the UGT71 B family) from Isatis indigotica can catalyze the production of phloridzin from phloretin in vitro. UGT236 shares high identity with P2'GT from apple. In this study, the recombinant plasmid pET28 a-MBP-UGT236 was transferred into Escherichia coli Rosetta(DE3) cells and induced by isopropyl-β-D-thiogalactoside(IPTG). The purified UGT236 protein was used for enzymatic characterization with phloretin as substrate. The results showed that UGT236 had the optimal reaction temperature of 40 ℃ and the optimal pH 8(Na_2HPO_4-NaH_2PO_4 system). The UGT236 activity was inhibited by Ni~(2+) and Al~(3+), enhanced by Fe~(2+), Co~(2+), and Mn~(2+), and did not affected by Mg~(2+), Ca~(2+), Li~+, Na~+, or K~+. The K_m, K_(cat), and K_(cat)/K_m of phloretin were 61.03 μmol·L~(-1), 0.01 s~(-1), and 157.11 mol~(-1)·s~(-1)·L, and those of UDPG were 183.6 μmol·L~(-1), 0.01 s~(-1), and 51.91 mol~(-1)·s~(-1)·L, respectively. The possible active sites were predicted by homologous modeling and molecular docking. By mutagenisis and catalytic activity detection, three key active sites, Glu391, His15, and Thr141, were identified, while Phe146 was related to product diversity. In summary, we found that the lignan glycosyltransferase UGT236 from I.indigotica could catalyze the reaction of phloretin into phloridzin. Several key amino acid residues were identified by structure prediction, molecular docking, and site-mutagenesis, which provided a basis for studying the specificity and diversity of phloretin glycoside products. This study can provide a reference for artificially producing glycosyltransferase elements with high efficiency and specific catalysis.
Glucosyltransferases/genetics* ; Glycosyltransferases/metabolism* ; Isatis ; Lignans/metabolism* ; Molecular Docking Simulation ; Phloretin/metabolism* ; Phlorhizin/metabolism*

Objective: Isoflavones and lignans are phytoestrogens that have recently gained interest as dietary factors related to prostatic diseases. However, no data on the concentrations in prostate tissue in humans is available. Therefore, the concentrations of isoflavones and lignans in plasma and prostatic tissues according to the prostate volume were compared to determine their possible effect on the benign prostatic growth. Methods: Fasting plasma and prostatic tissue specimens were acquired from 25 men over 50 years of age with similar normal dietary habits and no previous history of drug intake that could affect the isoflavones and lignans levels. The tissue was acquired either during a transurethral resection of the prostate in 15 patients with benign prostatic hyperplasia (BPH) with prostate volume over 40 ml or during a radical cystoprostatectomy in 10 patients with bladder cancer with a prostate volume < 25 ml, who were used as the controls. Quantitative analysis of the isoflavones, specifically equol, daidzein and genistein and lignans, particularly enterodiol and enterolactone, was performed by gas chromatography-mass spectrometry. Results: The mean prostatic concentrations of enterodiol, enterolactone, equol and daidzein in the BPH and the control groups were similar. However, the mean prostatic concentration of genistein was significantly lower in the BPH group than in the control group (65.43 +/- 17.05 vs 86.96 +/- 37.75 ng/ ml, respectively, p=0.032). The plasma concentration of isoflavones and lignans in the two groups were comparable. Conclusion: Isoflavones, but not lignans, have some influence the benign prostatic growth, and the prostatic concentration of genistein possibly has the closest association among them. More studies to further clarify the roles and mechanisms of isoflavone action on BPH including pharmacokinetic studies are recommended.
Blood/*metabolism ; Comparative Study ; Human ; Isoflavones/*metabolism ; Lignans/*metabolism ; Male ; Middle Age ; Osmolar Concentration ; Prostate/*metabolism ; Prostatic Hyperplasia/*metabolism ; Reference Values

OBJECTIVETo determine the plasma protein binding rate of arctiin and arctigenin.

METHODThe ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins.

RESULTThe plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively.

CONCLUSIONThe binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

Animals ; Binding, Competitive ; Blood Proteins ; metabolism ; Chromatography, High Pressure Liquid ; Furans ; metabolism ; Glucosides ; metabolism ; Humans ; Lignans ; metabolism ; Male ; Protein Binding ; Rats ; Rats, Sprague-Dawley ; Ultrafiltration ; methods

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor on hypoxia responses in mammalian tissues. HIF-1 plays as a positive factor in solid tumor and leads to hypoxia-driven responses that enhance its downstream gene expression for tumor growth and survival. LXY6099 was obtained by the structural modification and optimization of manassantin A (MA) as a high potent HIF-1 inhibitor. Antitumor activity of LXY6099 was observed in this study. LXY6099 with an IC50 value of 2.46 x 10(-10) mol x L(-1) showed more sensitive inhibition activity to HIF-1 than that of MA detected by reporter gene assay (> 100 folds). It showed strong inhibition on the growth of human solid tumor cell lines. Furthermore, LXY6099 exhibited significant antitumor activity against established human tumor xenografts in nu/nu mice with treatment of MX-1 breast cancer. Thus, LXY6099 as a novel HIF-1 inhibitor could be further developed into anti-cancer agents.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Heterografts ; Humans ; Hypoxia-Inducible Factor 1 ; metabolism ; Lignans ; pharmacology ; Mice, Nude

OBJECTIVETo investigate the protective effect of sesamin against cadmium chloride (CdCl2)-induced cardiotoxicity in rats.

METHODSFifty male Wistar rats were randomly assigned to five groups: control group, CdCl2 group, and low-, middle-, and high-dose sesamin groups. The control group was given normal saline. The CdCl2 group and sesamin groups were intraperitoneally injected with CdCl2 (5 mg/kg×2 d), and the low-, middle-, and high-dose sesamin groups were given 20, 40, and 80 mg/kg sesamin, respectively. All treatments lasted for four weeks. ECG was measured by a physiological recorder, and serum myocardial enzyme levels were determined by biochemical assay. The heart was weighed, and heart tissues were used in histopathological examination and determination of malondialdehyde (MDA) level.

RESULTSCompared with the control group, the CdCl2 group showed significantly higher levels of serum CK and CK-MB, an increased heart coefficient, significant ST-segment elevation, and higher level of MDA in myocardial tissue (P < 0.05). Histopathological analysis showed edema of myocardial tissues and cells, myocardial fibers disorder, karyopyknosis, and uneven or deep staining of nuclear chromatin. Different doses of sesamin relieved the myocardial pathological changes induced by CdCl2, and high-dose sesamin was the most effective. The middle- and high-dose sesamin groups showed significantly reduced serum CK and CK-MB levels compared with the CdCl2 group (P < 0.05). The heart coefficient of the high-dose sesamin group (0.19±0.01%) was significantly lower than that of the CdCl2 group (0.21±0.01%) (P < 0.05). Myocardial MDA levels of the three sesamin groups (42.32±4.65, 36.71±5.34, and 33.12±4.62 nmol/mg pro, respectively) were all significantly lower than that of the CdCl2 group (55.87±3.65 nmol/mg pro) (P < 0.05).

CONCLUSIONSesamin can relieve myocardial injury induced by CdCl2, and one possible mechanism is the enhancement of antioxidant capacity of myocardial tissue.

Animals ; Cadmium Chloride ; toxicity ; Creatine Kinase, MB Form ; blood ; Dioxoles ; pharmacology ; Heart ; drug effects ; Lignans ; pharmacology ; Male ; Malondialdehyde ; metabolism ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Wistar

OBJECTIVETo study the liposoluble ingredients of Quchiling (LQ), which enter the blood and the brain,and to confirm the active ingredients of LQ in vivo.

METHODSerum pharmacochemistry and gas chromatography mass spectroscopy were used to analyze ingredients of LQ entering the blood and the brain.

RESULTThere were eleven ingredients of LQ to enter the blood and six ingredients of LQ to enter the brain.

CONCLUSIONIt is confirmed that eleven ingredients of LQ entered the blood, which are beta-asarone, schisandrol A, schisandrol B, deoxyschisandrin, schisandrin B, schisantherrin A, schisantherrin B, schisantherrin C, delta-cadinene, delta-cadinol and calamendiol in the blood, and that six ingredients are beta-asarone, schisandrol A, schisandrol B, deoxyschisandrin, schisandrin B and calamendiol in the brain.

Animals ; Anisoles ; chemistry ; metabolism ; Brain ; metabolism ; Cyclooctanes ; chemistry ; metabolism ; Dioxoles ; chemistry ; metabolism ; Drugs, Chinese Herbal ; chemistry ; metabolism ; pharmacokinetics ; Female ; Gas Chromatography-Mass Spectrometry ; Lignans ; chemistry ; metabolism ; Male ; Polycyclic Compounds ; chemistry ; metabolism ; Rats ; Rats, Sprague-Dawley

AIMTo investigate the enzyme kinetics of (-)3S,4R,5R,6S-clausenamide[(-)-Clau] and (+)-3R,4S,5S,6R-clausenamide[(+)-Clau] catalyzed by rat liver microsomes and compare their stereoselective differences.

METHODSAn in vitro metabolic system was built by using rat liver microsomes and NADPH-generating system. Clau and its metabolites were determined simultaneously by a reversed-phase high performance liquid chromatography. The kinetic parameters, K(m), Vmax, and metabolic rate, Vmax/K(m), were calculated by Eadie-Hofstee plot.

RESULTSIn the metabolic system, (-)-Clau was found to be mainly metabolized to 7-hydroxy-, 5-hydoxy- and 4-hydroxy-Clau, and 7-hydroxylation was a preferential pathway which exhibited higher Vmax/K(m) value (0.135 microL.min-1.mg-1) than those of 5- and 4-hydroxylation (0.063 and 0.068 microL.min-1.mg-1, respectively). For (+)-Clau, it was mainly metabolized to 4-hydroxy-Clau, whereas 7-hydroxy- and 4-hydroxy-Clau were so small that they could not be detected systematically. 4-Hydroxylation of (+)-Clau showed highest Vmam/K(m) value (0.547 microL.min-1.mg-1) among all the metabolites tested, which was 8.0 times higher than that of 4-hydroxylation of its antipode.

CONCLUSIONThe data indicated that there were obvious substrate stereoselective differences in the hydroxylation metabolism of (+)- and (-)-Clau, which provided an explanation of the difference of pharmacokinetic characteristics of Clau enantiomers in rats.

Animals ; Drugs, Chinese Herbal ; isolation & purification ; metabolism ; Female ; Hydroxylation ; Kinetics ; Lactams ; chemistry ; isolation & purification ; metabolism ; Lignans ; chemistry ; isolation & purification ; metabolism ; Microsomes, Liver ; metabolism ; NADP ; metabolism ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Rutaceae ; chemistry ; Stereoisomerism

The effect of plant growth regulator forchlorfenuron (CPPU) 1 x 10(-6), 0.67 x 10(-6), 0.5 x 10(-6) on fruit morphology and effective components lignans was studied. Those morphologies were the combination of four basic morphological changes. The result showed, diametre were increased and longitudinal diametre of fruits were inhibited by foliage fertilizers including CPPU. At the same time, 1 000-grain weight and yield showed the varying degrees increase under CPPU. The order of the degree was 0.5 x 10(-6) > 1 x 10(-6) > 0.67 x 10(-6). Six lignans content of Schisandra chinensis of different harvest time and different CPPU processing groups were determined, the results showed that lignans accumulation occurred mainly in periods of premature the half mature fruiting stages. Under the 0.67 x 10(-6) CPPU treatment, schisandrol B, schisandrin B, schisandrin C content of S. chinensis showed different increase.
Chromatography, High Pressure Liquid ; Cyclooctanes ; analysis ; metabolism ; Dioxoles ; analysis ; metabolism ; Dose-Response Relationship, Drug ; Fruit ; drug effects ; growth & development ; metabolism ; Lignans ; analysis ; metabolism ; Phenylurea Compounds ; pharmacology ; Polycyclic Compounds ; analysis ; metabolism ; Pyridines ; pharmacology

There is some evidence that phytostrogen plays an important role in bone metabolism in postmenopausal women. In this study, we investigated the phytoestrogen intake levels and the relation between dietary phytoestrogens intake and bone mineral density of vegetarian (n = 77) and omnivore postmenopausal women (n = 122). Vegetarian women, all of them were seven day adventists, who had been on vegetarian diet (almost lacto-ovo vegetarians;a few vegans) over 20 yrs. The average age of vegetarians and omnivores were 62.3 yrs and 60.2 yrs, respectively and, there was no significant difference. However, body weight (p < 0.001), body mass index (p < 0.001) of vegetarians were significantly lower than those of omnivores. The mean daily energy intake of vegetarians and omnivores were 1386.1 kcal (76.3% of RDA) and 1424.5 kcal (76.9% of RDA), respectively. The mean calcium intake of vegetarians (456.7 mg, 66.3 % of RDA) was not significantly different from that of omnivores (453.5 mg, 65.2 % of RDA). The mean daily isoflavones (daidzein + genistein) intake of vegetarians and omnivore were 33.9 mg and 23.9 mg, respectively. The vegetarians consumed significantly greater quantities of isoflavones (p < 0.05) and lignans precursor (p < 0.05). In the vegetarians, intake of isoflavones was significantly positively correlated with BMD of femoral neck, after adjusted for age and BMI. Also lignan precursor intake of vegetarians was significantly positively correlated with BMD of spine, after adjusted for age and BMI. In conclusion, in omnivore post menopausal women, intake of phytoestrogen such as isoflavones and lignans was little low, and it is not clear that positive association with bone mineral density. But in vegetarian postmenopausal women, phytoestrogen intakes be important factors related to bone mineral density.
Body Mass Index ; Body Weight ; Bone Density* ; Calcium ; Diet, Vegetarian ; Energy Intake ; Female ; Femur Neck ; Humans ; Isoflavones ; Lignans ; Metabolism ; Phytoestrogens ; Spine

OBJECTIVETo study the metabolic chemistry and pharmaco-dynamics characters of ligan from seeds of Arctium lappa.

METHODHPLC method was used in the study. The analysis was carried out on C18 column. The mobile phase was CH3CN-0.05% H3PO4 (36:64) with flow-rate at 0.6 mL x min(-1) and wave-length of 210 nm. The column temperature was kept at 25 degrees C.

RESULTThe results indicated that the ligan was detected in plasma and the main organs 5 min after po. The main metabolic production in plasma was arctigenin. In addition, arctigenin and an unknown product were found in metabolic production in the organs.

CONCLUSIONThe method was stable,simple and reproducible. It can be used to determine the metabolic product of the ligan. The metabolic chemistry of ligan in plasma was obviously different from that in the main organs.

Animals ; Arctium ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Furans ; blood ; metabolism ; Glucosides ; blood ; metabolism ; Lignans ; blood ; isolation & purification ; metabolism ; pharmacokinetics ; Liver ; metabolism ; Male ; Mice ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Seeds ; chemistry ; Tissue Distribution