Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

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.     

Synthesis of morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) by dipeptidyl aminopeptidase IV derived from Aspergillus oryzae

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)), tetrapeptide, was synthesized using dipeptidyl aminopeptidase IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was incubated with Phe-Pro-NH(2) in the presence of DP IV under various conditions of temperature, concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40% yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt.HCl and 20 mM Phe-Pro-NH(2).HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4 degrees C for 24 h. Amino group protection was unnecessary for synthesis of morphiceptin by DP IV.     

Acylated and non-acylated flavonol monoglycosides from the Indian minor spice Nagkesar (Mammea longifolia)

A methanol extract of nagkesar (buds of Mammea longifolia), which showed strong radical scavenging activity, yielded 13 compounds by separations using column chromatography and HPLC. Structure elucidation of these compounds was achieved by (1)H and (13)C NMR, including DQF-COSY, TOCSY, DEPT, HMQC, HSQC, and HMBC. They include two new compounds, quercetin 3-O-(2' ',4' 'di-E-p-coumaroyl)-alpha-L-rhamno-pyranoside and quercetin 3-O-(3' ',4' '-di-E-p-coumaroyl)-alpha-L-rhamnopyranoside, along with known compounds kaempferol, quercetin, the isopropylidenedioxy derivative of shikimic acid, kaempferol 3-O-(2' ',4' '-di-E-p-coumaroyl)-alpha-L-rhamnopyranoside, kaempferol 3-O-(3' ',4' '-di-E-p-coumaroyl)-alpha-L-rhamnopyranoside, kaempferol 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, shikimic acid, kaempferol 3-O-beta-D-glucopyranoside, quercetin 3-O-beta-D-glucopyranoside, and beta-sitosterol 3-O-beta-D-glucopyranoside.     

Ingestion of water-soluble soybean fiber prevents osteopenia and hypercholesterolemia induced by ovariectomy in rats

This study examined the effects of feeding water-soluble soybean fiber (WSSF), a highly fermentable dietary fiber with low viscosity, on intestinal calcium (Ca) absorption, bone mineral content, and serum total cholesterol levels using female Sprague-Dawley rats (5 weeks old) with or without ovariectomy (OVX). The rats in each group were fed a WSSF (50 g/kg of diet) or WSSF-free diet for 4 weeks. Ca absorption was higher in OVX rats fed the WSSF diet than in OVX rats fed the WSSF-free diet. Femoral Ca content in both sham and OVX rats fed the WSSF diet was higher than that in rats fed the WSSF-free diet. The serum total cholesterol levels were elevated after OVX compared with that in the sham rats, and this elevation was prevented by the WSSF diet. In conclusion, ingestion of WSSF prevented osteopenia and hypercholesterolemia induced by ovariectomy in rats.     

Relationship between antibacterial activity of (+)-catechin derivatives and their interaction with a model membrane

(+)-Catechin derivatives with different alkyl chain lengths were synthesized from (+)-catechin and various straight chain alkylaldehydes in the presence of methyl mercaptan, and their antibacterial activities against Gram-positive bacteria were evaluated. The antibacterial activity increased markedly with elongation of the alkyl chain lengths of the derivatives and reached a maximum at a chain of four to seven carbons. Subsequently, interaction of the (+)-catechin derivatives with a model membrane using liposome was investigated. The derivatives with a chain of three carbons or more were found to have very strong affinity for the membrane. The injury action of the derivatives against the membrane was examined with liposome in which calcein was enclosed as a fluorescent indicator. The leakage was observed in the derivatives with chain lengths of four carbons or more. Particularly the derivatives with chains longer than five carbons are considered to destroy the liposome membrane judging from the degree of the fluorescent leakage. These results implied that the lipophilicity and disrupting ability of the (+)-catechin derivatives to the liposome membrane participate in their antibacterial activity.     

Precipitation chemistry in Japan 1989–1993

Precipitation chemistry in Japan was discussed on a wet-only sample database obtained in a nationwide survey from April 1989 to March 1993. Wet-only samples were collected at 29 stations over Japan on a biweekly basis. Commonly determined chemical parameters were measured in laboratories. The volume-weighted annual mean pH at each site ranged from 4.50 to 5.83 with a mean of 4.76. Concentration ranges and means (parenthesized) on an equivalent basis for major ions were as follows: nss-SO₄ ^2–; 5.2–58.9 (38.6), NO₃ ^–; 1.8–25.0 (14.1), NH₄ ⁺; 0.55–29.8 (18.3), nss-Ca²⁺; 2.0–34.5(14.2), Na⁺; 6.4–275.3 (49.1), Cl^–; 13.7–322.4 (63.5) eq L^–1. Acid-base relationships for Phase-II records were quantitatively discussed in terms of three measures: pH, fractional acidity, and our proposed pA_i.     

Long-Term Observation of Fogwater Composition at two Mountainous Sites in Gunma Prefecture, Japan

We made serial observations on acid fog at Mt. Akagi and Mt. Haruna, Japan for 5 years. The altitudes of the sampling sites were 1500 m (Mt. Akagi) and 1200 m (Mt. Haruna) above sea level, and the sites were approximately 30 km apart. The average liquid water content (LWC) at Mt. Akagi and Mt. Haruna was 74 mg m^?3 and 63 mg m^?3, respectively. The pH of fogwater was 2.72–7.14 (mean 3.71) at Mt. Akagi and 2.94–6.58 (mean 3.73) at Mt. Haruna. Our long-term observations indicate that there was no significant difference in the chemical components in fogwater at both sites except for ammonium ion. However, there were some cases where the chemical components of fogwater at each site were differed remarkably even in the concurrent fog event. Nitrate and sulfate ions contributed to acidification of fogwater at Mts. Akagi and Haruna and 95% of sulfate ion in the fogwater originated from air pollution. Ammonia gas in the air was the main neutralizer of acidity in fogwater. When absorption of excessive nitric acid gas over ammonia gas in the air occurred, the pH of fogwater was lowered. Our back trajectory analysis indicated that the fogwater at Mt. Akagi was mainly affected by an air mass from the Kanto Plain, including Tokyo, while the fogwater at Mt. Haruna was influenced by an air mass from large, western cities, such as Nagoya and Osaka, as well as Tokyo.     

Forecast of Sulfur Deposition in Japan for Various Energy Supply and Emission Control Scenarios

In order to contribute to the analysis and solution of regional scale environmental problems in East Asia, we developed a tool for the comprehensive assessment of alternative policy options to improve air quality. This tool projects the future regional energy supply, calculates the emission levels of sulfur dioxide and estimates the geographical pattern of sulfur deposition resulting from emissions. Sulfur deposition in Japan through 2030 was forecasted for various energy supply and emission control scenarios using the analysis tool. Future sulfur depositions were calculated from the source-receptor matrix for 1995 and the growth rate of emission for the source subregion. In the case of the current legislation scenario, anthropogenic SO₂ emissions in East Asia would grow by 34 percent and sulfur deposition in Japan would increase by approximately 20 percent between 1995 and 2030. This increase in sulfur deposition over these 35 years is sligthly less than the contribution from volcanic emission to sulfur deposition in Japan. In the case of the hypothetical dirty scenario for China, sulfur deposition in several grids which face the Sea of Japan would double by 2030.