Discovery of hyaluronidase inhibitors from natural products and their mechanistic characterization under DMSO-perturbed assay conditions.

The present study discovered four novel hyaluronan-degrading enzyme (hyaluronidase) inhibitors including chikusetsusaponins and catechins through the activity-guided separation of Panax japonicus and Prunus salicina, respectively. Although the discovery resulted in identification of usual frequent hitters, subsequent mechanistic characterizations under our DMSO-perturbed assay conditions and related protocols revealed that chikusetusaponin IV would serve as an aggregating and non-specific binding inhibitor, while (-)-epicatechin would interact specifically with enzyme at the catalytic site or more likely at a kind of catechin-binding site with a relatively week inhibitory activity. The latter description might provide a possible explanation for the well-known fact that a series of catechin have been described as frequent hitters in biological assays with a moderate activity. Thus, the present study demonstrated a practical and robust methodology to characterize initial screening hits mechanistically molecule-by-molecule in the early stage of natural product-based drug discovery.

Effects of eugenol-reduced clove extract on glycogen phosphorylase b and the development of diabetes in db/db mice.

We found that the 50% aqueous EtOH extract of clove (Syzygium aromaticum) had potent dose-dependent inhibitory activity toward glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes. Among the components, eugeniin inhibited glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes, with IC50 values of 0.14 and 4.7 µM, respectively. In sharp contrast, eugenol showed no significant inhibition toward glycogen phosphorylase b, even at a concentration of 400 µM. Eugenol-reduced clove extracts (erCE) were prepared and when fed to a db/db mouse they clearly suppressed the blood glucose and HbA1c levels. Furthermore, plasma triglyceride and non-esterified fatty acid levels in 5% and 10% erCE-fed db/db mice were significantly lowered, compared with control db/db mice without erCE supplementation. These results suggested that dietary supplementation with the erCE could beneficially modify glucose and lipid metabolism and contribute to the prevention of the progress of hyperglycemia and metabolic syndrome.

Antioxidant, free radical-scavenging activity and cytotoxicity of different solvent extracts and their phenolic constituents from the fruit hull of mangosteen (Garcinia mangostana).

Antioxidative, skin protective activities, and cytotoxicity of three extracts (water, methanol, and hexane) from the fruit hull of mangosteen (Garcinia mangostana Linn. (Guttiferae)) and their phenolic constituents such as alpha-mangostin, epicatechin, and tannin, were evaluated. The amounts of alpha-mangostin, total flavonoid, and total tannin were different among the three extracts, except those of total tannin in methanol and hexane extracts. For the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical scavenging, hydroxyl radical-scavenging, and inhibition of lipid peroxidation experiment, the water extract showed higher activity than the methanol extract and hexane extract. alpha-Mangostin, epicatechin, and tannin also revealed these antioxidant and free radical-scavenging activities. When added simultaneously with H(2)O(2) (200 microM) to keratinocyte cells, the water extract (50 microg/mL), epicatechin (200 microM), and tannin (200 microM) effectively protected cells from oxidative damage, but the methanol extract, hexane extract, and alpha-mangostin did not. The methanol extract and hexane extract exhibited moderate cytotoxicity, whereas alpha-mangostin showed strong cytotoxicity. The present study provides the evidence that Garcinia mangostana extracts, especially the G. mangostana water extract, act as antioxidants and cytoprotective agents against oxidative damage, which is at least partly due to its phenolic compounds in mangosteen.

2,5-Dideoxy-2,5-imino-d-altritol as a new class of pharmacological chaperone for Fabry disease.

Chromatographic separation of the extract from roots of Adenophora triphylla resulted in the isolation of two pyrrolidines, six piperidines, and two piperidine glycosides. The structures of new iminosugars were elucidated by spectroscopic methods as 2,5-dideoxy-2,5-imino-d-altritol (DIA) (2), beta-1-C-butenyl-1-deoxygalactonojirimycin (8), 2,3-dideoxy-beta-1-C-ethyl-1-deoxygalactonojirimycin (9), and 6-O-beta-d-glucopyranosyl-2,3-dideoxy-beta-1-C-ethyl-1-deoxygalactonojirimycin (10). beta-1-C-Butyl-1-deoxygalactonojirimycin (7) and compound 8 were found to be better inhibitors of alpha-galactosidase than N-butyl-1-deoxygalactonojirimycin. The present work elucidated that DIA was a powerful competitive inhibitor of human lysosome alpha-galactosidase A (alpha-Gal A) with a K(i) value of 0.5muM. Furthermore, DIA improved the thermostability of alpha-Gal A in vitro and increased intracellular alpha-Gal A activity by 9.6-fold in Fabry R301Q lymphoblasts after incubation for 3days. These experimental results suggested that DIA would act as a specific pharmacological chaperone to promote the smooth escape from the endoplasmic reticulum (ER) quality control system and to accelerate transport and maturation of the mutant enzyme.

Protective effects of dietary chamomile tea on diabetic complications.

Matricaria chamomilla L., known as "chamomile", has been used as an herbal tea or supplementary food all over the world. We investigated the effects of chamomile hot water extract and its major components on the prevention of hyperglycemia and the protection or improvement of diabetic complications in diabetes mellitus. Hot water extract, esculetin (3) and quercetin (7) have been found to show moderate inhibition of sucrase with IC50 values of 0.9 mg/mL and 72 and 71 microM, respectively. In a sucrose-loading test, the administration of esculetin (50 mg/kg body weight) fully suppressed hyperglycemia after 15 and 30 min, but the extract (500 mg/kg body weight) and quercetin (50 mg/kg body weight) were less effective. On the other hand, a long-term feed test (21 days) using a streptozotocin-induced rat diabetes model revealed that the same doses of extract and quercetin showed significant suppression of blood glucose levels. It was also found that these samples increased the liver glycogen levels. Moreover, chamomile extract showed potent inhibition against aldose reductase (ALR2), with an IC50 value of 16.9 microg/mL, and its components, umbelliferone (1), esculetin (3), luteolin (6), and quercetin (7), could significantly inhibit the accumulation of sorbitol in human erythrocytes. These results clearly suggested that daily consumption of chamomile tea with meals could contribute to the prevention of the progress of hyperglycemia and diabetic complications.

Iminosugars from Baphia nitida Lodd.

Chromatographic separation of the 50% aqueous EtOH extract of the leaves of the African medicinal tree Baphia nitida resulted in isolation of 10 iminosugars. The plant contained 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) as a major alkaloid. The structure of a new alkaloid was also elucidated by spectroscopic methods as the 1-O-beta-D-fructofuranoside of DMDP, and this plant produced 3-O-beta-D-glucopyranosyl-DMDP as well. DMDP is a potent inhibitor of beta-glucosidase and beta-galactosidase, whereas the other two derivatives lowered inhibition toward both of these enzymes and improved inhibitory activities toward rice alpha-glucosidase and rat intestinal maltase.

Isolation of glycosidase-inhibiting hyacinthacines and related alkaloids from Scilla socialis.

An examination of the bulbs of Scilla socialis has resulted in the isolation of 11 hyacinthacines, two pyrrolidines, and three piperidines. The structures of the new alkaloids were elucidated by spectroscopic methods as beta-1-C-ethyldeoxymannojirimycin (5), hyacinthacines B7 (10), C2 (11), C3 (12), C4 (13), and C5 (14), and alpha-5-C-(3-hydroxybutyl)hyacinthacine A2 (15). Although, beta-l-homofuconojirimycin (3) and alpha-7-deoxyhomonojirimycin (alpha-7-deoxy-HNJ, 4) are previously known alkaloids, this is the first report of their occurrence in the plant family Hyacinthaceae. Alkaloid 11 was found to be a good inhibitor of bacterial beta-glucosidase and human placenta alpha-l-fucosidase, with IC50 values of 13 and 17 microM, respectively, while alkaloid 12 showed no inhibitory activity toward alpha-l-fucosidase but was a more potent inhibitor of bovine liver beta-galactosidase (IC50 = 52 microM) than 11. Alkaloids 13 and 14 were shown to be inhibitory toward mammalian alpha-glucosidase (IC50 = 45 and 77 microM, respectively), and alkaloid 14 was demonstrated as a moderate inhibitor of bacterial beta-glucosidase (IC50 = 48 microM).

Inhibition of P-glycoprotein-mediated transport by terpenoids contained in herbal medicines and natural products.

Terpenoids form a large and structurally diverse family of natural products and are ingredients of various herbal medicines. We have investigated possible interactions between herbal medicines and conventional medicines, and recently reported that monoterpenoids contained in Zanthoxyli Fructus can be potent inhibitors of P-glycoprotein (P-gp). In the present study, the influence of 70 kinds of terpenoids present in natural products on P-gp-mediated efflux transport was investigated. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used to screen the terpenoids. Large increases in the intracellular accumulation of [(3)H]digoxin were observed in the presence of (R)-(+)-citronellal, (S)-(-)-beta-citronellol, alpha-terpinene, terpinolene, (-)-beta-pinene, abietic acid, ophiobolin A, cucurbitacin I, and glycyrrhetic acid. A study of the concentration-dependency revealed that the IC(50) of ophiobolin A, glycyrrhetic acid, (R)-(+)-citronellal, abietic acid, and cucurbitacin I was smaller than that of verapamil. The transcellular transport of [(3)H]digoxin across Caco-2 cell monolayers was then examined in the presence of (R)-(+)-citronellal, abietic acid, and glycyrrhetic acid. Significant increases in the apical-to-basolateral transport and decreases in the basolateral-to-apical transport and efflux ratio were demonstrated. These findings suggest that some natural products containing these terpenoids may inhibit P-gp-mediated transport and interact with P-gp substrates in the intestinal absorption process.

Inhibitory effects of Zingiber officinale Roscoe derived components on aldose reductase activity in vitro and in vivo.

Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Scientific research has gradually verified the antidiabetic effects of ginger. Especially gingerols, which are the major components of ginger, are known to improve diabetes including the effect of enhancement against insulin-sensitivity. Aldose reductase inhibitors have considerable potential for the treatment of diabetes, without increased risk of hypoglycemia. The assay for aldose reductase inhibitors in ginger led to the isolation of five active compounds including 2-(4-hydroxy-3-methoxyphenyl)ethanol (2) and 2-(4-hydroxy-3-methoxyphenyl)ethanoic acid (3). Compounds 2 and 3 were good inhibitors of recombinant human aldose reductase, with IC50 values of 19.2 +/- 1.9 and 18.5 +/- 1.1 microM, respectively. Furthermore, these compounds significantly suppressed not only sorbitol accumulation in human erythrocytes but also lens galactitol accumulation in 30% of galactose-fed cataract rat model. A structure-activity relationship study revealed that the applicable side alkyl chain length and the presence of a C3 OCH3 group in the aromatic ring are essential features for enzyme recognition and binding. These results suggested that it would contribute to the protection against or improvement of diabetic complications for a dietary supplement of ginger or its extract containing aldose reductase inhibitors.

Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli fructus.

Citrus (rutaceous) herbs are often used in traditional medicine and Japanese cuisine and can be taken concomitantly with conventional medicine. In this study, the effect of various citrus-herb extracts on P-glycoprotein (P-gp)-mediated transport was examined in vitro to investigate a possible interaction with P-gp substrates. Component monoterpenoids of the essential oil in Zanthoxyli fructus was screened to find novel P-gp inhibitors. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used. Cellular accumulation of [3H]digoxin was measured in the presence or absence of P-gp inhibitors or test samples. Aurantii fructus, Evodiae fructus, Aurantii fructus immaturus, Aurantii nobilis pericarpium, Phellodendri cortex, and Zanthoxyli fructus were extracted with hot water (decocted) and then fractionated with ethyl acetate. The cell to medium ratio of [3H]digoxin accumulation increased significantly in the presence of the decoction of Evodiae fructus, Aurantii nobilis pericarpium, and Zanthoxyli fructus, and the ethyl acetate fraction of all citrus herbs used. The ethyl acetate fraction of Zanthoxyli fructus exhibited the strongest inhibition of P-gp among tested samples with an IC50 value of 166 microg/mL. Then its component monoterpenoids, geraniol, geranyl acetate, (R)-(+)-limonene, (R)-(+)-linalool, citronellal, (R)-(+)-citronellal, DL-citronellol, (S)-(-)-beta-citronellol, and cineole, were screened. (R)-(+)-citronellal and (S)-(-)-beta-citronellol inhibited P-gp with IC50 values of 167 microM and 504 microM, respectively. These findings suggest that Zanthoxyli fructus may interact with P-gp substrates and that some monoterpenoids with the relatively lower molecular weight of about 150 such as (R)-(+)-citronellal can be potent inhibitors of P-gp.