Tohru Mitsunaga

Inhibitory Effects of Oolong Tea Polyphenols on Pancreatic Lipase in Vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.

Comparison of Antifungal and Antioxidant Activities of Acacia mangium and A. auriculiformis Heartwood Extracts

The effect of heartwood extracts from Acacia mangium (heartrot-susceptible) and A. auriculiformis (heartrot-resistant) was examined on the growth of wood rotting fungi with in vitro assays. A. auriculiformis heartwood extracts had higher antifungal activity than A. mangium. The compounds 3,4′,7,8-tetrahydroxyflavanone and teracacidin (the most abundant flavonoids in both species) showed antifungal activity. A. auriculiformis contained higher levels of these flavonoids (3.5- and 43-fold higher, respectively) than A. mangium. This suggests that higher levels of these compounds may contribute to heartrot resistance. Furthermore, both flavonoids had strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and laccase inhibition. This suggests that the antifungal mechanism of these compounds may involve inhibition of fungal growth by quenching of free radicals produced by the extracellular fungal enzyme laccase.

Termite repellent sesquiterpenoids from Callitris glaucophylla heartwood

Fractions of methanol and ethanol extracts from the heartwood of white cypress pine (Callitris glaucophylla Thompson et Johnson) were investigated for their repellent activity against subterranean termite Coptotermes formosanus Shiraki worker using a two-choice semicircular filter paper test at 0.5% (w/w) concentration. Fraction CY-E2 composed of (−)-citronellic acid, guaiol, α-, β-, and γ-eudesmol isomers as well as an unknown compound, showed the highest statistically significant repellency (97.8% ± 2.2 SEM) of all fractions tested. Bioactivity-guided fractionations using high-performance liquid chromatography led to the isolation of two, oxygenated eudesmane-type sesquiterpenes with α-methylene moieties, both termite-repellent compounds. These compounds were subsequently identified as ilicic acid methyl ester (IAME) and costic acid by means of spectroscopic analyses, electron impact mass spectrometry, and nuclear magnetic resonance spectroscopy. We report the isolation of both IAME and costic acid from C. glaucophylla heartwood for the first time.

Tyrosinase inhibitory activity of proanthocyanidins from woody plants

Flavanol values, molecular weight distributions, polyphenolic patterns, and tyrosinase inhibitions of proanthocyanidins (PACs) from karamatsu (Larix sp.) bark, acacia Morishima (Acacia mearnsii) bark, and commercial quebracho (Schinopsis lorentzii) extracts were examined to investigate the relation between the structures of PACs from woody plants and tyrosinase inhibitory activities. Aqueous acetone (70%) extracts of acacia showed high flavanol contents and strong tyrosinase inhibition. On the other hand, the quebracho extracts inhibited tyrosinase activity slightly despite its high flavanol content. Phenyl nucleus analysis by the nucleus exchange reaction provided information that karamatsu, quebracho, and acacia extracts consist of procyanidin, profisetinidin, and prorobinetinidin, respectively. The relation between the phenolic hydroxylation pattern and tyrosinase inhibition suggested that the PACs with a 5,7-dihydroxyphenyl structure in the A-ring and a 3,4,5-trihydroxyphenyl structure in the B-ring have potent tyrosinase inhibitory activity.

Color development of proanthocyanidins in vanillin-hydrochloric acid reaction

The influence of proanthocyanidin (PA) structures contained in bark on color development in the vanillin-hydrochloric acid (V-HCl) method used widely as a quantitative method for measuring PA were examined. The maximal absorption wavelength was different in terms of the bark from which the PA was obtained. Phenyl nucleus (resorcinol, phloroglucinol) constituting the A-ring of PA reacts with vanillin to produce the color. The maximal absorption wavelengths of the solutions from synthesized procyanidin and profisetinidin were 500 and 540 nm, respectively, indicating that the color tone differs in the V-HCl method based on the hydroxylation patterns of the A-ring. The colored solution of (+)-catechin with vanillin was dialyzed, and the resulting product (C-VC) was analyzed by gel permeation chromatography and 1H nuclear magnetic resonance. It was found that C-VC was a polymer complex consisting of 9mol (+)-catechin moieties and 10mol vanillin moieties. It was presumed that the cationized vanillin molecules that do not combine with (+)-catechin play an important role on color development in the presence of C-VC.

Inhibitory Effects of Bark Proanthocyanidins on the Activities of Glucosyltransferases of Streptococcus Sobrinus

Abstract Seventy percent acetone aqueous extracts of Acacia mearnsii and Larix spp. bark inhibited the activity of glucosyltransferase (GTase) derived from Streptococcus sobrinus to a greater degree than green tea or oolong tea extracts. The GTase inhibitory activity increased as the molecular weight of proanthocyanidins contained in the extracts increased. The GTase inhibition was very closely related to the hydroxylation patterns in the B-rings of synthesized proanthocyanidins, where compound with a catechol ring showed high activity than with a pyrogallol B-ring, and it was a non-competitive type of inhibition.

In vitro screening of angiotensin I-converting enzyme inhibitors from Japanese cedar (Cryptomeria japonica)

Screening and isolation of angiotensin I-converting enzyme (ACE) inhibitors from Japanese cedar (Cryptomeria japonica) based on the in vitro ACE inhibitory assay were attempted. The ethanol extract from outer bark showed the highest inhibitory activity (IC50 is 16μg/ml) among 24 extracts prepared from roots, leaves, heartwood, sapwood, inner bark, and outer bark by successive extraction with four solvents. The fractionation of the outer bark ethanol extract followed by the bioassay resulted in the isolation of two strong ACE inhibitors, catechin and dimeric procyanidin B3. The bioassay of three flavan-3-ols including (+)-catechin and six flavones revealed that most of these compounds have high ACE inhibitory activity. The results suggest that the phenolic hydroxyl group at the C7 position and heterocyclic oxygen atom of these compounds are important for expressing the inhibitory activity.

Polyphenols in Acacia mangium and Acacia auriculiformis heartwood with reference to heart rot susceptibility

The heartwood of Acacia mangium is vulnerable to heart rot and this is the first study to investigate the role of heartwood extractives in its susceptibility. Acacia auriculiformis was compared with A. mangium because it is rarely associated with heart rot. The heartwood extracts of both species were dominated by three flavonoids (2,3-trans-3,4′,7,8-tetrahydroxyflavanone, teracacidin, and 4′,7,8,-trihydroxyflavanone), which were purified and identified by nuclear magnetic resonance spectroscopy. The latter compound has not been previously reported in A. mangium and evidence for melacacidin is also newly reported. The mass spectrometric (MS) behavior of these compounds is given, for example teracacidin does not form molecular ions by either electrospray ionization or atmospheric-pressure chemical ionization. The nature of Acacia tannins was compared to quebracho tannin (composed of profisetinidins) using oxidative cleavage to enable MS detection but a negative reaction was obtained for both, which suggests the Acacia tannins may also be of the 5-deoxy proanthocyanidin type. The concentration of flavanones was less when A. mangium heartwood was decayed but the amount of proanthocyanidins was only slightly reduced and therefore these compounds may be more resistant to degradation by heart rot fungi. We found that the total phenol content of A. auriculiformis was about fivefold that of A. mangium, and, while preliminary, this provides evidence for a role played by phenolic extractives in heart rot resistance of these Acacia species.

Investigating antitermitic compounds from Australian white cypress heartwood (Callitris glaucophylla Thompson et Johnson) against Coptotermes formosanus Shiraki

Abstract Antitermitic activities of extracts obtained from the heartwood of Australian white cypress (Callitris glaucophylla Thompson et Johnson) were investigated in an attempt to detect and identify the presence of biologically active compounds against Coptotermes formosanus Shiraki. Extraction of compounds was achieved by successive extraction using dichloromethane and methanol. Three sesquiterpene alcohols, guaiol, α-eudesmol and β-eudesmol and two monoterpene acids, (−)-citronellic acid and geranic acid, were found in the C. glaucophylla heartwood extracts based on high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analysis. Antitermitic activity of each fraction obtained from methanol extracts was demonstrated by repellent and no-choice feeding tests. One of the strong termiticidal active compounds identified was columellarin and another sesquiterpene lactone fraction showed repellent activity.

Predicting the hydroxymethylation rate of phenols with formaldehyde by molecular orbital calculation

The rates (k) of hydroxymethylation of phenol, resorcinol, phloroglucinol, and several methylphenols in diluted 10% dimethylformamide aqueous alkaline solution were calculated based on the consumption of phenols and formaldehyde. Thek values of phloroglucinol and resorcinol relative to that of phenol were about 62000 and 1200 times, respectively. The phenols that have methyl or hydroxyl groups at the C-3 or C-5 position (or both) have larger rate constants than phenols with substituents at other positions. Several kinds of atomic charge of the carbons on the aromatic ring of phenols were calculated using the semiempirical orab initio method. The correlations between the averagek (Ave.k) and average electrostatic charges (Ave.q) at the carbons were fairly good. Highest occupied molecular orbitals (HOMO) were observed. The best correlation between Ave.k and Ave.q was obtained when diphenols and triphenols were assumed to exist in solution as their respective di-anion.