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We aimed to develop a new food-processing approach using pectin to reduce gastrointestinal absorption of mycotoxins. When Ca2+ is added to low-methoxyl pectin, a gel resembling an egg box-like structure forms that is able to trap certain molecules. We examined whether or not low-methoxyl amidated pectin (LMA) and low-methoxyl non-amidated pectin (LMNA) trapped the mycotoxin deoxynivalenol (DON) after being ingested. We first determined the trapping effects of LMA and LMNA on DON in vitro under conditions similar to those in the human stomach, with results showing that LMA gel trapped DON to a greater extent than the LMNA gel. We then performed in vivo experiments and demonstrated that the LMA gel containing DON reduced DON's absorption from the gastrointestinal tract. This new food-processing technique holds great promise for reducing the bioavailability of DON in contaminated food and may be useful in mitigating the effects of other mycotoxins.

Deoxynivalenol (DON) and nivalenol (NIV), trichothecene mycotoxins, are secondary metabolites produced by Fusarium fungi. Trichothecene mycotoxins cause immune dysfunction, thus leading to diverse responses to infection. The present study evaluated the effect of DON and NIV on nitric oxide (NO) production by RAW264 cells stimulated with lipopolysaccharide (LPS). LPS-induced NO production was reduced in the presence of these toxins. The transcriptional activation and expression of inducible NO synthase (iNOS) by LPS were also repressed by these toxins. DON or NIV inhibited LPS-induced expression of interferon-beta (IFN-beta), which plays an indispensable role in LPS-induced iNOS expression. These results indicate that DON and NIV inhibit the LPS-induced NO and IFN-beta production, which both play an important role for host protection against invading pathogens, and suggests that the inhibition of these factors may be involved in the immunotoxic effects of these mycotoxins.

An organic solvent-tolerant monoclonal antibody specific to aflatoxins B(1), B(2), G(1), G(2), and M(1) (AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1)) was prepared. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) values were 1.9, 2.1, 2.1, 2.4, and 2.8 ng/mL for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1), respectively. Antibody reactivity was retained at 40% methanol concentration or at acetonitrile concentrations up to 40%. An immunoaffinity column (IAC) was prepared using agarose gel beads with bound antibody. The IAC retained the tested AFs that were 89, 90, 95, 90, and 89% for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1) at 20% acetonitrile concentrations or that were 81, 87, 79, and 83% for AFB(1), AFB(2), AFG(1), and AFG(2) at 60% methanol concentrations. Roasted peanuts and seven kinds of spices were spiked with 8.0, 1.0, 6.0, and 1.0 ng for AFB(1), AFB(2), AFG(1), and AFG(2) per 1 g sample and extracted with 90% acetonitrile. The roasted peanuts and cayenne pepper out of the spices were also extracted with 70% methanol. The extracts were diluted 5-fold with phosphate-buffered saline and applied to the IAC. The spiked aflatoxins were recovered with satisfactory rates: 78 (RSD, 2.1%) to 127% (RSD, 1.7%). The developed IAC was used for the analysis of aflatoxins in naturally contaminated samples of roasted peanuts and cayenne pepper. The newly developed IAC showed substantially organic solvent tolerance at the concentration that could not be used for existing IACs, and the column showed good ability to clean up samples for food analysis.

The mean concentration and daily intake of inorganic food additives (nitrite, nitrate, and sulfur dioxide), antioxidants (BHA and BHT), a processing agent (propylene glycol), and a sweetener (sodium saccharin) were estimated based on the results of an analysis of 34,489 food samples obtained in official inspections by 106 local governments in Japan in fiscal year 1998. The ratios of mean concentrations of these seven food additives to each allowable limit were 20.0%, 53.9%, 15.5%, 6.2%, 0.4%, 18.5%, and 5.7%, respectively. The daily intakes of these food additives estimated from their concentrations in foods and the daily consumption of foods were 0.205, 0.532, 4.31, 0.119, 0.109, 77.5, and 7.27 mg per person, respectively. These amounts were 6.8%, 0.3%, 12.3%, 0.5%, 0.7%, 6.2%, and 2.6% of the acceptable daily intake (ADI), respectively, when body weight was assumed to be 50 kg. No remarkable differences in the daily intakes of these seven food additives or the ratios to the ADI were observed compared with the results based on the official inspections in fiscal years 1994 and 1996.

The daily intake of 106 food additives (measured as 43 compounds) by five age groups (small children, children, youths, adults, and aged) was estimated. The estimation was based on the concentrations of the compounds in the food mixtures of seven categories consisting of 246 foods by the market basket method. The daily intake of most food additives increased with age. The daily intake of xylitol, however, was approximately ten times higher in small children and children than in the aged. It was clearly shown that the daily intake of food additives differed among the age groups, and that the differences are attributable to different food consumption patterns. None of the food additives exceeded the ADI, except nitrate, whose intake was 1.1–2.2 times higher than the ADI. The source of most of the nitrate was food in category VII (fruits, vegetables, and seaweed) where nitrate is mostly present as a natural component and the benefit of fruit and vegetable intake is well known as stated by JECFA.

To clarify toxic effects of long-term oral administration of low dose cadmium (Cd) on the liver and kidney, six groups of female Sprague-Dawley rats were fed a diet containing Cd-polluted rice or CdCl2 at concentrations up to 40 ppm, and killed after 12, 18, and 22 months. With toxicological parameters, including histopathology, there was no evidence of Cd-related hepato-renal toxicity, despite a slight decrease of mean corpuscular volume and mean corpuscular hemoglobin of red blood cells with 40 ppm CdCl2. Dose-dependent accumulation of Cd was observed in the liver and kidneys with peak levels of 130 +/- 42 micrograms/g and 120 +/- 20 micrograms/g, respectively, at 18 months in animals treated with 40 ppm CdCl2. A dose-dependent increase in urinary Cd levels became evident with time. Induction of metallothionein (MT) was also observed in the liver and kidney with a high correlation to the corresponding Cd levels. In the proximal renal tubular epithelia of 40 ppm CdCl2-treated rats at 22 months, prominent accumulation of Cd was observed in secondary lysosomes associated with MT deposits in their exocytotic residual bodies. The results demonstrated that, in contrast to the case with high-dose Cd-administration, renal toxicity is not induced by long-term oral administration of low amounts of Cd, although tissue accumulation does occur. Possible protective mechanisms may be operating.

Samples of umeshu, a Japanese plum liqueur made from unripe plums, shochu and crystal sugar, were stored under fluorescent light, in the dark and in the refrigerator. The amount of ethyl carbamate formed in umeshu exposed to light or room temperature was larger than that in the dark or at low temperature. The amount of ethyl carbamate formed in umeshu to which cyanide had been added was larger than that in the absence of added cyanide. Thus, the amount of ethyl carbamate formed in the umeshu was increased by not only light and higher temperature, but also cyanide. Samples of model alcoholic beverages were stored under various conditions using red, yellow and blue cellophanes. The amount of ethyl carbamate formed in the model alcoholic beverage with blue cellophane was larger than in the cases of red and yellow cellophanes. It was found that the amount of ethyl carbamate formed in the model alcoholic beverage was increased by light in the wavelength range of 375-475 nm.

The stability and degradation pathways of barbaloin (BA) in aqueous solution were investigated over the pH range of 1.1-8.4. The experiments were performed at three different temperatures (5, 20 and 50°C) by the use of high performance liquid chromatography. The degradation of BA was observed as a pseudo first-order kinetics and shown to proceed via racemization (Keq) and degradation processes (K2), (Keq>K2). The racemization product, isobarbaloin (isoBA), further underwent the same degradation as did BA. 10-hydroxyaloins A (10-HA), 10-hydroxyaloins B (10-HB) and aloe-emodin (AE) were detected as the degradation products of BA and isoBA. On the basis of kinetic studies, it was found that the degradation rate of BA decreased with decreasing pH and temperature. The half-lives of BA at pH7.2 (20°C) and pH3.4 (20°C) were 1.15day and 5.00day, respectively. The present results are useful for the integrity of foods containing aloe.

Alkyl p-hydroxybenzoates such as isobutyl p-hydroxybenzoate (PHBA-iBu), butyl p-hydroxybenzoate (PHBA-nBu), isopropyl p-hydroxybenzoate (PHBA-iPr), propyl p-hydroxybenzoate (PHBA-nPr), ethyl p-hydroxybenzoate (PHBA-Et), and methyl p-hydroxybenzoate (PHBA-Me) are widely used as preservatives, stabilizers and antiseptics for medical supplies, cosmetics, foodstuffs etc. We determined the binding affinity of alkyl p-hydroxybenzoates to human estrogen receptor alpha (ER alpha) and beta (ER beta) by non-RI receptor binding assays. PHBA-iBu had a high binding affinity for ER alpha (IC50: 6.0 x 10(-6) M, the relative binding affinity (RBA): 0.267) and ER beta (IC50: 5.0 x 10(-6) M, RBA: 0.340). These IC50 values and RBA were almost the same as those of bisphenol A. The ranking of the estrogenic potency of alkyl p-hydroxybenzoates for both ERs is different; that is, PHBA-iBu > PHBA-nBu[symbol: see text]PHBA-iPr[symbol: see text]PHBA-nPr > PHBA-Et >> PHBA-Me. Alkyl p-hydroxybenzoates bound with equal relative affinity to both ER alpha and beta proteins. Alkyl p-hydroxybenzoate having a long alkyl side-chain showed a high affinity for ER alpha and beta. These findings suggest that p-hydroxybenzoates may be endocrine disruptors.

Previously, we reported that KS-F2 (non-dialyzable fraction of hot water extracts from skin of Aloe arborescensMiller) and AS-F2 (non-dialyzable fraction of hot water extracts from skin of Aloe africanaMiller) with de-protein showed a high mitogenic activity towards cells of mice (_??_) (C3HeN/C3HeJ). The water-soluble polysaccharides obtained were fractionated and purified by ion-exchange chromatography on a DEAE-Toyopearl 650M column. The chemical structres of K-1, the mitogenic activity component of KS-F2 and A-1, those of AS-F2, were analyzed. Arabinose and galactose were contained in both K-1 and A-1. The molar ratio of arabinose to galactose was 1 : 1 in K-1, and 1 : 2 in A-1 before reduction and 1 : 3 in K-1, and 1 : 3 in A-1 after reduction. The ratio of galactose increased both K-1 and A-1. Consequently, uronic acid seemed to be galacturonic acid. The ratio of neutral sugar to uronic acid contents in K-1 and A-1 was about 1 : 1 and 5.6 : 1, respectively. K-1 and A-1 were found as gel-filtration fractions with a high molecular weight of approximately 1, 700, 000 and 1, 750, 000, respectively. The main components in K-1 were found to be arabinose and galactose based on their [α] D value of +127 (H2O) and a peak of 890 in IR ν max cm-1. A-1 also showed arabinose and galactose based on their [α] D value of-33 (H2O) and a peak of 830 in IR ν max cm-1. Therefore, K-1 seemed to be a β-glucosidic bond, and A-i was close to a β-glucosidic bond. After the controlled hydrolysis of K-1 and A-1, an investigation of the bond form was performed using thin layered chromatography. It was suggested that arabinose and uronic acid were located on the outside and that arabinose and galactose constituted the core of the K-1 and A-1 structures. Using GC/MS, the methylation analysis gave 1, 4-di-O-acetyl-2, 3, 5-tri-O-methyl arabitol, 1, 5-di-O-acetyl-2, 3, 4, 6-tetra-O-methyl galactitol, 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl arabitol, 1, 3, 5-tri-O-acetyl-2, 4, 6-tri-O-methyl galactitol, 1, 5, 6-tri-O-acetyl-2, 3, 4-tri-O-methyl galactitol and 1, 2, 5, 6-tetra-O-acetyl-3, 4-di-O-methyl galactitol in the molar ratios of 4.3 : 3.4:1.0:3.1:1.9:6.0 from the K-1 core, and 2.4:2.5:1.0:3.8:1.1:2.8 from the A-1 core, respectively.

To investigate the relationship between cadmium (Cd) toxicity, intestinal absorption, and its distribution to various tissues in rats treated orally with minimum amounts of Cd, 14 female rats per dose group per time point were given diets consisting of 28% purified diet and 72% ordinary rice containing Cd-polluted rice (0. 02, 0.04, 0.12, or 1.01 ppm of Cd) or CdCl(2) (5.08, 19.8, or 40.0 ppm of Cd) for up to 8 months. At 1, 4, and 8 months after the commencement of Cd treatment, seven rats per group were euthanized for pathological examinations to determine the Cd concentrations in the liver and kidneys and metallothionein (MT) in the liver, kidneys, intestinal mucosa, serum, and urine. One week before each period of 1, 4, and 8 months, the remaining seven rats in each group were administered a single dosage of (109)Cd, a tracer, to match the amounts of the designated Cd doses (about 1.2 to 2400 microg/kg body wt). They were euthanized 5 days later to determine the distribution of Cd to various tissues. No Cd-related toxic changes were observed. The concentrations of Cd in the liver and kidneys at any time point and MT in the liver, kidney, serum, and urine at 4 and 8 months increased dose-dependently, whereas MT in the intestinal mucosa did not alter markedly at any time point. The distribution rates of Cd to the liver increased dose-dependently (40% at lower doses to 60% at higher doses), whereas those to the kidney decreased dose-dependently (20% at lower doses to 10% at higher doses). The Cd retention rates 5 days after (109)Cd administration (amounts of Cd in various tissues/amounts of Cd administered) ranged from 0.2 to 1. 0% at any time point. These results suggest that the distribution of Cd to the liver and kidneys after the oral administration vary depending on the dosage levels of Cd. The difference of the distribution pattern of Cd to the liver and kidney is probably due to the difference in the form of the absorbed Cd, i.e., free ion or Cd-MT complex, although not closely related to the MT in the intestinal mucosa.

Mitogenic activity and antitumor activity of Aloe arborescens Miller, Aloe barbadensis Miller and Aloe africana Miller, which have been used as traditional drugs and health food, were studied. The raw fresh aloe was divided into flesh and skins, and was lyophilized. Fraction were obtained by extraction with hot water, 0.1M NaOH and 0.5M NaOH. Mitogen assay was performed for all fraction using spleen cells from mice (_??_), C3H/HeN and C3H/HeJ. Six fractions showed mitogenic activity towards spleen cells of C3H/HeN. In addition, although the proteins were removed with actinase, the samples continued to show this activity. Among them, the non-dialyzable fraction of hot water extracts from a skin of Aloe arborescens Miller and the non-dialyzable fraction of hot water extracts from a skin of Aloe africana Miller showed strong mitogenic activity towards cells of C3H/HeJ, suggesting that it was free from bacterial endotoxic lipopolysaccharide. These results suggest that the activity components are not located in the protein region. No fraction showed antitumor activity against Sarcoma 180 ascites in mice.

Previously, we have reported on the mitogenic activity of Aloe arborescens Miller, Aloe barbadensis Miller, and Aloe africana Miller. Six fractions, the non-dialyzable fraction of hot water extracts (KS-F2) from the skin of Aloe arborescens Miller, the dialyzable fraction of hot water extracts (BF-F1) from the flesh of Aloe barbadensis Miller, the dialyzable fraction of hot water extracts (AF-F1), the non-dialyzable fraction of 0.1 M NaOH extracts (AF-F3), From the flesh of Aloe africana Miller, and the non-dialyzable fraction of hot water extracts (AS-F2), and the non- dialyzable fraction of 0.1M NaOH extracts (AS-F3) from the skin of Aloe africana Miller, showed mitogenic activity towards cells from mice (_??_), C3H/HeN and C3H/HeJ. In this paper, we report on some properties of polysaccharide fractions from which mitogenic activity was shown. A polysaccharide obtained from KS-F2 and AS-F2 which showed strong mitogenic activity with de protein was fractionated and purified by ion-exchange chromatography by DEAE-Toyopearl 650 M. Each of two fractions from KS-F2 mainly contained neutral sugar, and uronic acid. All of three fractions from AS-F2 contained neutral sugar, and one of them contained uronic acid. These results of mitogen activity assays for the major fractions suggested that the activity components of AS-F2 were located in the fraction containing uronic acid, and those of AS-F2 were located in the fraction that had greater amounts of polysaccharides, such as neutral sugar and uronic acid, than the other fractions. A polysaccharide obtained from six fractions was examined regarding the components and molar ratio of sugar by gaschromatography. Its fraction were composed of large amounts of arabinose and galactose.

Four alkaloids, 5′-des- O -methylharringtonine, 3′ S -hydroxy-5′-des- O -methylharringtonine, 5′-des- O -methylhomoharringtonine and 5′-des- O -methylisoharringtonine, were isolated from the leaves and stems of C. harringtonia var. drupacea. Their structures were established by 1 H and 13 C NMR spectroscopic studies, chemical conversions to known alkaloids and applying the advanced Mosher's method for the secondary alcohol moiety.

9-Anthroylnitrile was used as an achiral reagent for the derivatization of carnitine. The reagent forms UV-absorbing derivatives with the hydroxyl groups of carnitine enantiomers under very mild conditions. The derivatives were separated by high-performance liquid chromatography on an ovomucoid-conjugated column with a mobile phase of acetonitrile-20 mM KH 2 PO 4 (adjusted to pH 4.5 with phosphoric acid) (17:83, v/v). The separation factor (α) and resolution ({ce:inline-formula}R s{/ce:inline-formula}) of the enantiomers were 1.44 and 5.05, respectively. The calibration plots indicated good linearity over a sample concentration ranging from 0.2 to 1.0 mg ml −1, and the detection limit at 254 nm was 0.05 mg ml −1 for each carnitine enantiomer. The reproducibility in the analysis of 1 mg ml −1 of each enantiomer was within 2.0%. The method was applied successfully to the determination of carnitine enantiomers in pharmaceutical preparations.

Polyclonal antibodies against P-1, a pectic polysaccharide fraction extracted with 0.5 M NaOH from the kernels of Prunus mume and consisted of arabino-galacturonan, and I-3, the partial acid (0.1 M trifluoroacetic acid) hydrolysate of P-1, were prepared in Japanese white rabbits. Competitive ELISA experiments strongly suggested that anti P-1 and anti I-3 antibodies were different but P-1 and I-3 cross-reacted with each other to recognize a partly similar epitope structure. The reactivities of polysaccharide fractions from the raw flesh of P. mume, and the kernels of apricot and peach extracted with either water or sodium hydroxide were examined using both antisera by the indirect competitive ELISA method. The polysaccharide fractions extracted with sodium hydroxide solutions had the reactivities but not those extracted with cold and hot water. These facts suggested that the similar structure of polysaccharides to P-1 was present in the flesh of P. mume and the kernels of apricot and peach. However, neither pectin of apple nor citrus had reactivity with each antiserum. P-1 would be different in chemical structure from a commercially available pectin, a water-soluble polysaccharide from apple and citrus.

Three oxygenated cephalotaxine ester-type alkaloids, 11α-hydroxyhomodeoxyharringtonine (1), 11β-hydroxyhomodeoxyharringtonine (2) and 11β-hydroxydeoxyharringtonine (3) were isolated from Cephalotaxus harringtonia var. drupacea. Their structures were established by spectroscopic methods including 2D NMR and CD spectra, and their antileukemic activity was evaluated using P-388 leukemia cells.

An analytical procedure was developed for the determination of total bromide residues in crude drugs and their preparations, using modified X-ray fluorescence spectrometry. A 0.26 g of powdered sample mixed with 2.34 g of cellulose powder as vehicles was applied to a 40 mm i.d. aluminum ring, then pressed for one minute and set in spectrometer. The limit of the determination was 1μg/g (10μg/g as the original sample). Using the described method, cellulose powder as vehicles offers an extremely good property for mixing samples and less interference of sample matrix. We were able to eliminate the previous procedure, which is necessary to prepare standards for each part of sample due to being liable to variation. The modified X-ray method is advantageous for simple and high speed measurements and could be applied for various kinds of crude drugs and their preparations.

P-1 was partially hydrolyzed with 0.01, 0.05, and 0.1 M trifluoroacetic acid (TFA), successively, and the dialyzable (E-1, E-2, and E-3) and non-dialyzable (I-1, I-2, and I-3) fractions were prepared and analyzed chemically and immunochemically. Either I-1 or E-1 reacted with anti P-1 serum as strongly as P-1 and were mitogenic. The cross-reactivity of I-2 and I-3 was less than I-1 with anti P-1 serum. However, they were as mitogenic as I-1. The cross-reactivity of E-2 and E-3 to anti P-1 serum was also very weak, and they were not mitogenic. The E-1 fraction had a similar sugar composition to I-1 and P-1. E-2 was a monosaccharide, all of Ara, and would be from the linkage of furanosyl residues in P-1. The composition of E-3 was free from Ara and the structure of E-3 was similar to that of I-3. E-3 would be considered to be deleted arabinofuranose from E-1. These results suggest that the mitogenic activity measured by the alkaline phosphatase assay is a property of the core part, I-3, but that P-1 contains several epitopes other than the core part by the immunochemical analysis.

Fourteen types of corona discharge needles made of different materials were used for high performance liquid chromatography/atmospheric pressure chemical ionizationquadrupole mass spectrometer (LC/APCI-QMS), and after a lapse of one hour and five hours from the start of the equipment, selected-ion monitoring determination of 3,5-di-tert-butyl-4-hydroxytoluene (BHT) was made seven times repeatedly. Suitability as a discharge needle was judged by the total (n=14) relative standard deviation (T-RSD) of the quantitative determination value. T-RSD's of the iron needle, tin-plated iron needle, silver alloy needle, platinum alloy needle and stainless steel needle were 1-3%.It could be elucidated from the scanning electron micrograph of the needle surface that suitability as a discharge needle was related to the stability of needle surface material against the corona discharge. The iron needle, having a small T-RSD was judged to be the most suitable as a corona discharge needle for quantitative analysis.

A polysaccharide fraction extracted with cold 0.5M NaOH from the kernels of Prunus mume exhlbited some biological activities. A polysaccharide, P-1, was purified from the 0.5M NaOH extract by ion-exchange chromatography and gel-flltration. The results of the structural analysis of P-1 to determine the relationship between the activities and the structure are described in this paper. In the mild acid hydrolysis of P-1, the nondialyzable hydrolysate (I-3) believed to be its core portion was obtained. The yield of I-3 was 26.0% and contained 59.8% uronic acid as galacturonic acid (GalA). The neutral sugars of I-3 were composed of rhamnose, xylose and galactose in a molar ratio of 1.0 : 3.4 : 0.3 following analysis by gas-liquid chromatography. The molecular weight of I-3 was estimated to be ca. 14000 by gel-filtration on Toyopearl HW55F. I-3 exhibited the mitogenic activity toward spleen cells as well as P-1. These facts appeared to confirm that I-3 was the core part of P-1 and important for its biological activity. I-3 was successfully reduced by the Taylor and Conrad method to avoid so much repetition. Methylation analysis of the reduced hydrolysate by gas-liquid chromatography and gas chromatography-mass spectroscopy showed that the ratio of 1, 4-linked galactopyranosyl and 1, 3, 4-linked galactopyranosyl residues were significantly increased in comparison with native I-3. These results suggested that I-3 was composed of 1, 4- and 1, 3, 4-linked galacturonic acid residues in the main chain.

Cephalotaxidine (1), a novel Cephalotaxus dimeric alkaloid, has been isolated from Cephalotaxus harringtonia var. drupacea, and its structure has been elucidated based on spectroscopic analyses. Alkaloid 1 has a unique CC bridge between the two mother skeletons which are composed of homoharrigtonine and its oxygenated congener.

Drupangtonine ( 1 ), a novel Cephalotaxus alkaloid, has been isolated from Cephalotaxus harringtonia var. drupacea , and its structure has been elucidated based on spectroscopic analyses. Compound 1 strongly inhibits the growth of P-388 leukemia cells.

A rapid coulometric method was developed for the measurement of peroxide value In edible oils and fats. The sample size and reagents volumes In this method are considerably less than those in the American Oil Chemists' Society method. Iodine produced by the reaction of the Iodide Ion and peroxide In the sample Is electrochemlcally reduced at the carbon-felt electrode more rapidly than it is with lodometric titration. The present method Is successfully applied to the measurements of edible oils and fats, and the coulometric results obtained are consistent with those obtained by iodometry.

Physicochemical properties and antitumor activities of a fungal (1----3)-beta-D-glucan, OL-2, isolated from Leiwan (Omphalia lapidescens) were examined. OL-2 showed sharp signals on carbon-13 nuclear magnetic resonance spectrum in dimethylsulfoxide-d6 as a solvent, and these signals were significantly reduced by the addition of distilled water to the concentration of 20%. This phenomenon is consistent with the general property of the gel forming (1----3)-beta-D-glucan. Binding of OL-2 to Congo red induced a significant change of lambda max to a longer wavelength, and the concentration to induce gel to sol transition was about 0.7 N; in contrast, the concentration was about 0.2 N in the cases of SPG and curdlan. These observations suggested that the gel structure would be significantly stabilized in the case of OL-2. OL-2 showed no or low antitumor activity against the solid form of Sarcoma 180 by intraperitoneal and intralesional administrations; however, it was effective on the ascites form of Sarcoma 180. Of interest, OL-2 also showed significant antitumor activity against the ascites form of MH-134 when administered with 5-fluorouracil. These results indicated that OL-2 showed characteristic features regarding its physicochemical properties and antitumor activity.

Activation of the alternative pathway of complement (APC) and the Limulus factor G cascede by an alkali-soluble glucan, OL-2, isolated from a crude drug "leiwan" (Omphalia lapidescens) and its Smith-type degradation products were examined using human whole complement and Limulus lysate. OL-2 is a (1→3)-β-D-glucan having two branches at every third main chain glucosyl unit at each C-6 position (degree of branching (DB) : 2/3), and DB of the derivatives used in this study were OL-2-I (1/3), OL-2-II (1/2), OL-2-III (1/24) and OR-OL-2c (no branch : 0/1). Activation of APC by these glucans was dependent on incubation time and concentraiton. Under optimal conditions, OL-2-I, OL-2-II and OL-2-III showed stronger activation of APC than did the origical OL-2, and OR-OL-2c showed relatively weak activity. The structure-activity relationship of the activation of the factor G cascade was similar to that of APC. Considering the fact that the less branched derivatives of OL-2 also showed significantly stronger antitumor activity than OL-2, too much branching would suppress the recognition of (1→3)-β-D-glucan by the host-self defecse mechanisms.

A method for the determination of bromine in food products by ion chromatography was developed. Three grams of sample was ashed in alkaline solution in a nickel crucible at 500°C. The ashed residue dissolved in deionized water was filtrated through a membrane filter and the filtrate was applied to ion chromatography. The eluted ions were monitored at 193 nm with a spectrophotometric detector. A chloride ion in 4500 fold excess had no interfering effect on the determination of a bromide ion. The recoveries from food products examined were 90.0-102%. The determination limit was 0.5 μg/g in food. The proposed method was found to be efficient for the determination of total bromine in foods.

A simple and rapid method for the analysis of inorganic phosphates used for the treatment of vegetables is described. Inorganic phosphates were extracted with water from the vegetable. The extract was mixed with Mo (V)-Mo (VI) color-producing reagent, and the mixture heated at 120-140°C for 10-15min in order to hydrolyze polyphosphates to orthophosphates. The resultant orthophosphates were allowed to react with the Mo (V)-Mo (VI) reagent to from a heteropoly blue complex. Inorganic phosphates were visually semi-quantitative in comparison with a standard solution. When the method was applied to commercial vegetables, it was found that ginger tuber was treated with phosphate more frequently than the other vegetables. This method was applicable to the quantitative analysis by measurement of absorbance at 820 nm.

A systematic method was developed for the analyses of 15 kinds of artificial dyes and 19 kinds of natural dyes in foods. Dyes were extracted from the sample with water and dichloromethane. Water soluble dyes in the aqueous extract were purified and fractionated into fractions I and II by use of polyamide. Oil soluble dyes in the dichloromethane extract were purified and fractionated into fractions III-V by liquid-liquid partition, silica gel column and alumina column chromatographies. The dyes in each fraction were identified by thin-layer chromatography. This method was applied for the detection of artificial and natural dyes in various commercial foods.

熱水抽出後の残渣を,0.1M水酸化ナトリウム,次いで0.5M水酸化ナトリウムで抽出した.抽出物は除タンパク後,それぞれゲルろ過による分画・精製を行ない,0.1M水酸化ナトリウム抽出物からはP-4を,0.5M水酸化ナトリウム抽出物からはP-5を得た.P-4及びP-5については,ゲルろ過,及び電気泳動により単一性を確認した.それぞれの旋光度及び赤外吸収スペクトルからβ優位の結合であることが推察された.P-4の分子量は約105000, P-5は約90000であった.P-4の構成糖は,mannose, galactose, glucoseであり,そのモル比は1.0:1.8:4.2であり,P-5はglucanであった.P-4は過ヨウ素酸酸化及びスミス分解,メチル化分析,部分酸加水分解の結果,glucoseの(1→3)結合の直鎖をcoreとし,glucose及びgalactoseの(1→6)結合の分枝を持ち,galactoseの非還元末端はmannoseであることを推定した.また,P-5は過ヨウ素酸酸化,スミス分解,メチル化分析の結果,glucoseの(1→3)結合の直鎖をcoreとし,(1→6)結合の短い枝のある構造であることを推定した.

シロタモギタケの熱水抽出画分を, Sephacryl S-400ゲルで分画したP-2及びP-3について,ゲルろ過,超遠心分析,電気泳動により均一物質であることを確認した後,構造解析を行なった.(1) 物理的性質は,旋光度〔a〕20D及び赤外吸収スペクトルの結果から, P-2及びP-3共にβ結合を優位とする構造であることがわかった.これらの平均分子量はP-2約108000, P-3約16000であった.(2) 構成糖については, P-2はマンノース,ガラクトース,グルコースから成り,そのモル比は1.0:2.3:1.4であり, P-3はマンノース,ガラクトースが1.0:3.1からなるグリカンであることがわかった.(3)過ヨウ素酸酸化,スミス分解,メチル化分析及び酸部分分解-メチル化分析の結果, P-2はマンノースの非還元末端,グルコース(1→3),ガラクトース(1→6),グルコース(1→3)(1→6),ガラクトース(1→2)(1→6)の比がおおむね3:2:4:1:2であることがわかった.以上, P-2はグルコースの(1→3)結合をcoreとしガラクトースの(1→6)結合の枝の中に,さらにガラクトースの(1→6)結合の枝を持ち,いずれも非還元末端にマンノースを有する構造であることが推察された.P-3はマンノースの非還元末端,ガラクトース(1→6),ガラクトース(1→2)(1→6)がおおむね1:2:1であり, P-3はガラクトースの(1→6)結合をcoreとし,ガラクトースの(1→6)結合の枝を持ち,いずれも非還元末端にマンノースを有する構造であることが推察された.

Antioxidative and synergistic activities with antioxidants of sucrose ester of fatty acids (SEF) were investigated with respect to autoxidation of lard. The degree of oxidation of lard was measured by using peroxide, acid, iodine and dinitrophenylhydrazine values. To examine stabilities of butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT) and tocopherols (Toc) added to lard, these antioxidants were determined by high performance liquid chromatography, respectively. Under autoxidative conditions, slight antioxidative and synergistic activities of SEF with BHA or Toc were observed. By the addition of SEF, stabilities of BHA or Toc in lard increased, whereas stability of BHT was similar to that without SEF.

シロタモギタケから抽出した水溶性多糖LU-1について精製及び分画を行ない, P-1, P-2, P-3の3つの画分を得た.今回はそれらのうちP-1について構造解析を行なった.(1) LU-1は約18%のタンパクを含有する他,ほとんどが中性糖で構成されていることが分かった.その構成糖はマンノース,ガラクトース,グルコースであり,モル比は1.0:1.9:2.6であった.(2) LU-1をプロナーゼで処理し,除タンパク後,Sephacryl S-400を用いたゲルろ過により, P-1, P-2,P-3の3つの画分を得た.これらのうちP-1はゲルろ過,電気泳動,超遠心分析により, P-1は均一物質であることを確認した.(3) P-1はマンノース,ガラクトース,グルコースが1:2:5のモル比で構成され,平均分子量約470000でβ結合を優位とするグリカンであり,分子内はグルコースの(1→3)結合をcoreとし,グルコースの非還元末端と,マンノースを非還元末端に持つガラクトースの(1→6)結合の分枝を持つ構造であることを推定した.

The cytotoxic alkaloids nordeoxyharringtonine (2), homodeoxyharringtonine (3), and bishomodeoxyharringtonine (4) were isolated from the leaves and stems of Cephalotaxus harringtonia var. drupacea. Their structures were established on the basis of spectral data and chemical evidence.

The mean concentrations and daily intake of four antifungal agents were estimated based on the results of an analysis of 7,005 samples of food obtained in official inspections by Japanese local governments in fiscal year 1998. The mean concentration of diphenyl was 0.0004% of the allowable limit, and those of imazalil, o-phenylphenol, and thiabendazole were 14.0%, 3.5%, and 5.7%, respectively. The daily intakes of these antifungal agents per person, estimated from their concentrations and the daily consumption of the foods, were 0.000326, 1.89, 11.5, and 23.3 micrograms, respectively, and assuming a body weight of 50 kg, the amounts of these antifungal agents consumed were 0.000013%, 0.15%, 0.12%, and 0.47% of the acceptable daily intake, respectively. These values are similar to the values obtained on the basis of the results of the official inspection in fiscal years 1994 and 1996, except that the amount of diphenyl is much lower (1/100).

Daily intake of isoflavones (daidzin, glycitin, genistin, daidzein, glycitein, and genistein) was determined quantitatively, based on the market basket method. Acid hydrolysis during extraction of foods was chosen to convert phytoestrogenes into the respective aglycons, facilitating HPLC analysis and allowing quantitation of total isoflavones as aglycones including both originally present glycosides and "free" aglycones. The isoflavones were extracted from samples with methanol and determined by reversed-phase HPLC analysis using a linear gradient of methanol-water as the eluent. From the results of hydrolysis, the daily intake of total isoflavon was 38.1 mg/adult Japanese. The values obtained by the market basket method and the National Nutrition Survey method were similar.

A simple and rapid method using capillary zone electrophoresis (CZE) for the determination of milt protein (MP), which contains mainly protamine, and polylysine (PL) in food additive preparations and processed foods was developed. CZE separation was performed on poly(vinyl alcohol)-coated capillaries at a column temperature of 20 degrees C with 120 mmol/L phosphate buffer (pH 2.5) as the running buffer. The influence of various components in food additive preparations on CZE analysis of MP and PL was examined. Egg white lysozyme, glycine, sodium acetate, glycerol, fumaric acid, calcium carbonate, dextrin, emulsifiers and sodium polyphosphate and pyrophosphate had no effect. No peak of protamine was detected in preparations containing metaphosphate. The analysis method for processed foods was composed of extraction with 4% formic acid, precipitation of macromolecular compounds with ethanol, concentration in a water bath and determination by CZE. The average recoveries were 108.4% for protamine sulfate (PS) in red bean sticky rice, and 81.3% for PL in white rice, 118% in egg sandwiches, and 115% in shiraae. The limits of detection of PS in red bean sticky rice and PL in white rice were both 50 ppm.

To determine whether low-dose oral administration of cadmium (Cd) induces renal toxicity, six groups of female Sprague-Dawley rats were fed a diet containing low amounts of CdCl2 or Cd-polluted rice at concentrations up to 40 ppm, and were killed after 12, 18, and 22 months (experiment 1). In addition to the determination of cortical Cd levels and histopathological assessment of kidneys, labeling indices (LIs) for proliferating cell nuclear antigen (PCNA) in the renal cortical tubular epithelium of Cd-treated rats were determined as a measure of regenerative activity. For comparison, the kidneys of rats given diets containing small to large amounts of CdCl2 up to 600 ppm for 4 months were similarly examined (experiment 2). Animals in experiment 1 demonstrated spontaneous chronic nephropathy and fluctuation in the tubular PCNA LI, but these findings were not correlated with renal Cd levels at 22 months. PCNA LI on the other hand, appeared to be linked to the severity of chronic nephropathy. In experiment 2, levels of CdCl2 of 200 ppm or more clearly induced degeneration and apoptosis of proximal tubules with high correlations between renal Cd levels, PCNA LI, and the severity of tubular degeneration. The results demonstrated that, in contrast to high-dose Cd administration, treatment with 40 ppm or less for 22 months did not influence tubular regeneration as a component of nonspecific chronic nephropathy, suggesting that long-term oral administration of low levels of Cd does not injure renal tubules in female rats.

Analytical data of Smith-type degradation products and carbon-13 nuclear magnetic resonance spectroscopy of an alkali-soluble glucan, OL-2, which was isolated from a crude fungal drug "Leiwan" (Omphalia lapidescens), suggested that OL-2 was a β-1, 3-D-glucan possessing approximately two branches at every three main chain glucosyl unit at each C-6 position. Antitumor activities of OL-2 and the degradation product, OR-OL-2c, against the solid form of sarcoma 180 in ICR mice were negative.

A sensitive and selective high-performance liquid chromatographic method with post-column derivatization, using o-phthalaldehyde (OPA) and 3-mercaptopropionic acid (3-MPA), is described for the determination of biotin in pharmaceutical preparations. The use of 3-MPA gives intense fluorescent derivative and improves the stability of biotin fluorophore towards oxidation to the picomole level. The fluorophore was detected at 453 nm (excitation at 342 nm). The calibration graph was linear for 20-200 ng per injection. The detection limit of biotin under these conditions was about 10 ng per injection. The RSDs were 1.9-3.4%. This method could be applied to pharmaceutical preparations without interference of other compounds.

Three alkaloids, neoharringtonine, homoneoharringtonine and 3'S-hydroxyneoharringtonine, were isolated from the leaves and stems of Cephalotaxus harringtonia var. drupacea. Their structures were established by spectroscopic methods, including two-dimensional NMR and CD spectra, and their antileukaemic activity was evaluated using P-388 leukaemia cells.

A method is described for the determination of carnitine chloride in pharmaceutical preparations by precolumn derivatization and high-performance liquid chromatography (HPLC). Carnitine chloride is derivatized by reaction with pyrene-1-carbonyl cyanide (PCC) in dimethyl sulphoxide. The PCC derivative of carnitine is then used for quantitative HPLC analysis on a cation-exchange column with fluorescence detection. The calibration graph was linear over a sample concentration range from 2.0 to 20 micrograms/ml; the limit of detection was 0.5 microgram/ml under the conditions studied. The proposed method was applied satisfactorily to the determination of carnitine chloride in pharmaceutical preparations.

The fruits of Prunus mume SIEB. et ZACC. (Japanese name, ume) have been used as a traditional drug and health food. In order to study the active components of P. mume, the polysaccharide fractions were extracted with cold water, hot water and aqueous sodium hydroxide from the kernels of P. mume. We found that some of the polysaccharide fractions exhibited various types of biological activities such as mitogenesis, activation of the alternative pathway of complement and activation of clot formation in human plasma. A polysaccharide, P-1, obtained from the cold 0.5 M NaOH extract was purified by ion-exchange chromatography and gel-filtration. P-1 contained 62.0% neutral sugar as glucose and 38.4% uronic acid (as galacturonic acid), and was free from protein. The neutral sugars of P-1 were arabinose, xylose, rhamnose and galactose in a molar ratio of 9.4 : 3.4 : 1.1 : 1.0, following analysis by gas-liquid chromatography. In addition, galacturonic acid was identified by thin-layer chromatography. The molecular weight of P-1 was found to be more than 2000000 by gel-filtration on Toyopearl HW 65F. P-1 showed mitogenic activity towards spleen cells of both C3H/HeN and C3H/HeJ, suggesting that it was free from bacterial endotoxic lipopolysaccharides.

The immunopharmacological activities of a fungal (1-->3)-beta-D-glucan, OL-2, isolated from "Leiwan" Omphalia lapidescens were examined. Intraperitoneal (i.p.) administration of OL-2 to ICR mice induced a significant number of peritoneal exudate cells (PEC) and white blood cells over the period of a few days. Spleen cell numbers were also increased by i.p. administration of OL-2 at about a week. These changes reverted to the normal level within a month. Responses of spleen cells and bone marrow cells (BM) to colony stimulating factors (CSF) were augmented by OL-2 administration assessed by cell proliferation assay. Sera from OL-2 administered mice contained an increased concentration of colony stimulating activity. Gene expressions of interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha in the spleen were also increased. These results suggested the activation of hematopoietic responses, and would well relate to the incremental increase in PEC, white blood cell and spleen cell numbers. OL-2 also increased the serum concentration of fibronectin and complement component C-3. However, OL-2 did not show adjuvant activity to SRBC and antitumor activity against the solid form of Sarcoma 180 by i.p. administration. Yet, OL-2 did not interfere with the antitumor activity of SSG against the same tumor system. These facts suggested that OL-2 could enhance nonspecific host defense mechanisms by enhancing hematopoietic responses, but would not enhance or inhibit the specific immunity mediated by lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)