Genistein-derivatives from Tetracera scandens stimulate glucose-uptake in L6 myotubes.

An EtOAc-soluble partition of the MeOH extract of a branch of Tetracera scandens (Dilleniaceae family) was subjected to a glucose-uptake assay, which led to the isolation and identification of five isoflavones of previously known structure namely, genistein (1), its derivatives 3',5'-diprenylgenistein (2), 6,8-diprenylgenistein (3), derrone (4) and alpinumisoflavone (5). Of these, compounds 2--5 exhibited significant glucose-uptake activity in basal and insulin-stimulated L6 myotubes. The findings from adenosine monophosphate-activated kinase (AMPK) activation and glucose transport protein4 (GLUT4) and GLUT1 over-expression revealed certain characteristics of compounds 2--5. These compounds inhibited protein tyrosine phosphatase 1B (PTP1B) activities with IC50 values ranging from 20.63 +/- 0.17 to 37.52 +/- 0.31 microM. No muscle cell toxicity was reported with compounds 3--5, while compounds 1 and 2 reduced muscle cell viability with IC50 values of 34.27 +/- 0.35 and 18.69 +/- 0.19 microM, respectively. It was concluded that T. scandens and its constituents exerted highly desirable activities on type 2 diabetes mellitus treatment since they significantly stimulated the uptake of glucose, AMPK phosphorylation, GLUT4 and GLUT1 mRNA expressions and PTP1B inhibition in L6 myotubes.

Anti-diabetic properties of chrysophanol and its glucoside from rhubarb rhizome.

An ethanol extract of rhubarb rhizome exhibited marked glucose transport activity in differentiated L6 rat myotubes. Activity-guided fractionation resulted in the isolation of two anthraquinones, chrysophanol-8-O-beta-D-glucopyranoside (1) and chrysophanol (2). The anti-diabetic effect was examined by glucose transport activity, glucose transporter 4 (Glut4) expression in myotubes, and the level of insulin receptor (IR) tyrosine phosphorylation as influenced by tyrosine phosphatase 1B, each of which is a major target of diabetes treatment. Chrysophanol-8-O-beta-D-glucopyranoside up to 25 microM dose-dependently activated glucose transport in insulin-stimulated myotubes. Increased tyrosine phosphorylation of IR due to tyrosine phosphatase 1B inhibitory activity with an IC50 value of 18.34+/-0.29 microM and unchanged Glut4 mRNA levels was observed following chrysophanol-8-O-beta-D-glucopyranoside treatment. Chrysophanol up to 100 microM exerted mild glucose transport activity and elevated the tyrosine phosphorylation of IR via tyrosine phosphatase 1B inhibition (IC50=79.86+/-0.12 microM); Glut4 mRNA expression was also significantly increased by 100 microM. The ED50 values of the two compounds were 59.38+/-0.66 and 79.69+/-0.03 microM, respectively. Therefore, these two anthraquinones from rhubarb rhizome, chrysophanol-8-O-beta-D-glucopyranoside and chrysophanol, have mild cytotoxicity and anti-diabetic properties and could play metabolic roles in the insulin-stimulated glucose transport pathway.

Structural importance of the acyl group in substrate specificity of purified bovine lysophospholipase D.

The structural importance of the acyl group in lysophosphatidylcholine (LPC) as substrate of purified bovine lysophospholipase D (lysoPLD) was investigated. Among LPCs with saturated acyl chains, the K(m) value decreased according to the length of the acyl chain (C12-C16) up to the palmitoyl group, while the V(m) value showed no remarkable change. But, the extension of the acyl size to C18, as observed with 1-stearoyl LPC (K(m), 8.5 mM), rather resulted in a remarkable increase in the K(m) value. Meanwhile, the introduction of one double bond in the C18 saturated acyl chain led to a remarkable reduction in the K(m) value, as observed with 1-oleoyl LPC (K(m), 0.48 mM). Furthermore, 1-linoleoyl LPC (K(m), 56 microM) with two double bonds exhibited a smaller K(m) value than 1-oleoyl LPC, suggesting that the unsaturation degree might be important in augmenting the binding affinity of LPCs. A similar phenomenon was also observed with 1-arachidonoly LPC (K(m), 79 microM) or 1-docosahexaenoyl LPC (K(m), 36 microM). Overall, the order of catalytic efficiency (V(m)/K(m) value) of those LPCs seemed to be affected by the K(m) value rather than the V(m) value, which differed by at most threefold among LPC derivatives. Next, the introduction of a hydroperoxide group into 1-linoleoyl-LPC or 1-arachidonoyl LPC led to a further reduction in K(m) values (1-hydroperoxylinoleoyl LPC, 26 microM; 1-hydroperoxyarachidonoyl LPC, 33 microM), accompanied by a further increase in the V(m)/K(m) values. Additionally, phosphatidylcholines (PCs) with an oxidized acyl chain at sn-2 position were found to be efficient as 1-palmitoyl LPC as substrates of lysoPLD. Taken together, the catalytic efficiency of LPCs or oxidized PCs as substrates of lysoPLD seems to be determined by the property of the acyl chain, length of the acyl chain, unsaturation degree and oxidation status.

Inhibition of lysophospholipase D activity by unsaturated lysophosphatidic acids or seed extracts containing 1-linoleoyl and 1-oleoyl lysophosphatidic acid.

Lysophospholipase D (lysoPLD), generating lipid mediator lysophosphatidic acid (LPA) from lysophosphatidyclcholine (LPC), is known to be inhibited by lysophosphatidic acids. Meanwhile, some plant lipids are known to contain lysophospholipids as minor components. Therefore, it is interesting to test whether edible seed samples, rich in phospholipids, may contain lysophospholipids, which express a strong inhibition of lysoPLD activity. First, the structural importance of fatty acyl group in LPAs was examined by determining the inhibitory effect of various LPAs on bovine lysoPLD activity. The most potent in the inhibition of lysoPLD activity was linoleoyl-LPA ( K i, 0.21 microM), followed by arachidonoyl-LPA ( K i, 0.55 microM), oleoyl-LPA ( K i, 1.2 microM), and palmitoyl-LPA ( K i, 1.4 microM), based on the fluoresecent assay. The same order of inhibitory potency among LPA analogs with different acyl chains was also found in the spectrophotometric assay. Subsequently, the extracts of 12 edible seeds were screened for the inhibition of lysoPLD activity using both spectrophotometric and fluorescent assays. Among seed extracts tested, the extract from soybean seed, sesame seed, or sunflower seed (30 mg seed weight/mL) was found to exhibit a potent inhibition (>80%) of lysoPLD activity. In further study employing ESI-MS/MS analysis, major LPA components in seed extracts were identified to be 1-linoleoyl LPA, 1-oleoyl LPA, and 1-palmitoyl LPA with 1-linoleoyl LPA being more predominant. Thus, the potent inhibition of lysoPLD activity by seed extracts might be ascribed to the presence of LPA with linoleoyl group rather than other acyl chains.

PTP1B inhibitory activity of kaurane diterpenes isolated from Siegesbeckia glabrescens.

Protein tyrosine phosphatase 1B (PTP1B) is considered as a therapeutic target for the treatment of diabetes and obesity. In our preliminary screening study, a MeOH extract of the aerial part of Siegesbeckia glabrescens was found to inhibit PTP1B activity at 30 microg/mL. Bioassay-guided fractionation led to the isolation of two active diterpenes, ent-16betaH, 17-isobutyryloxy-kauran-19-oic acid (1) and ent-16betaH, 17-acetoxy-18-isobutyryloxy-kauran-19-oic acid (2), along with ent- 16betaH, 17-hydroxykauran-19-oic acid (3). Compounds 1 and 2 inhibited the PTP1B activity with IC50 values of 8.7 +/- 0.9 and 30.6 +/- 2.1 microM, respectively. Kinetic studies suggest that both 1 and 2 are non-competitive inhibitors of PTP1B. However, compound 3 substituted with a hydroxyl group at C-17 in kaurane-type showed no inhibitory effects towards PTP1B.

Antidiabetic effects of extracts from Psidium guajava.

During a screening of medicinal plants for inhibition of protein tyrosine phosphatase1B (PTP1B), an extract from Psidium guajava (Myrtaceae) leaves exhibited significant inhibitory effect on PTP1B. Thus, its antidiabetic effect on Lepr(db)/Lepr(db) mice was evaluated. Significant blood glucose lowering effects of the extract were observed after intraperitoneal injection of the extract at a dose of 10mg/kg in both 1- and 3-month-old Lepr(db)/Lepr(db) mice. In addition, histological analysis of the liver from the butanol-soluble fraction treated Lepr(db)/Lepr(db) mice revealed a significant decrease in the number of lipid droplets compared to the control mice. Taken together, it was suggested that the extract from Psidium guajava leaves possesses antidiabetic effect in type 2 diabetic mice model and these effect is, at least in part, mediated via the inhibition of PTP1B.

A new VHR dual-specificity protein tyrosine phosphatase inhibitor from Dendrobium moniliforme.

Bioassay-guided fractionation of the EtOAc-soluble extract of Dendrobium moniliforme afforded a new phenanthraquinone-type metabolite, 7-hydroxy-5,6-dimethoxy-1,4-phenanthrenequinone ( 1), along with the previously reported 5-hydroxy-3,7-dimethoxy-1,4-phenanthrenequinone ( 2). The structures of the compounds were identified mainly on the basis of MS and NMR data. Compound 1 inhibited VHR dual-specificity protein tyrosine phosphatase (DS-PTPase) activity in a dose-dependent manner, displaying an IC (50) value of 3.0 +/- 0.2 microM.

Inhibition of VHR dual-specificity protein tyrosine phosphatase activity by flavonoids isolated from Scutellaria baicalensis: structure-activity relationships.

Three flavonoids: norwogonin, dihydronorwogonin and baicalein, were isolated from the roots of Scutellaria baicalensis, as potential inhibitors of VHR dual-specificity protein tyrosine phosphatase (DS-PTPase). Norwogonin (IC 50 = 1.1 microM), dihydronorwogonin (IC 50 = 2.9 microM) and baicalein (IC 50 = 2.4 microM) showed potent inhibitory activity toward VHR, but had no inhibitory activity against T-cell protein tyrosine phosphatase or serine/threonine protein phosphatase 1. From comparisons to the inhibitory activities of other similar flavonoids, it could be suggested that the presence of a hydroxy group in the B ring of flavonoids interferes with the inhibitory activity toward VHR DS-PTPase.

Expression and characterization of Ca(2+)-independent lipase from Bacillus pumilus B26.

A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M(r)) of 19,225. Based on the M(r) and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 degrees C and 8.5, respectively. The lipase B26 showed a 'Ca(2+)-independent thermostability and catalytic activity'. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca(2+)-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C(14)-C(18)) and triolein (C(18:1)) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.