It is well known that some species of phthalate esters have reproductive and developmental toxicity, monoesters being active metabolites. In June 2000, the Japanese Government established a tolerable daily intake (TDI) for di(2-ethylhexyl) phthalate (DEHP) based on data for testicular and reproductive toxicity because of the report that certain cooked foods had been relatively high contaminated, caused by the use of polyvinyl chloride gloves containing DEHP in the final stage of food packing. In the present paper, first of all, no observed adverse effect levels (NOAELs) or lowest observed adverse effect levels (LOAELs) of various kinds of phthalate esters in terms of reproductive and developmental toxicity endpoints, and the differences with age and species in testicular toxicity are examined and summarized. Then the rationale for establishing a TDI for DEHP and the exposure problem are discussed. This article also includes new information from a draft of the Toxicological Profile for DEHP published in September 2000 by the US Agency for Toxic Substances and Disease Registry, and assessment reports made available in October 2000 from the Center for the Evaluation of Risks to Human Reproduction, US National Toxicology Program.

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The inhibitory effect of guava leaf extract (GvEx) on influenza virus infection was examined using the hemagglutination-inhibition test and the preventive effect on influenza infection in mice. GvEx had the same level of hemagglutination-inhibition activity for two different subtypes (PR8 (H1N1) and Guizhou (H3N2)) of human influenza A virus. It was found that GvEx-treated influenza virus lost its infectivity so that there was decreased morbidity and increased survival of mice after nasal injection. Investigation of various canned tea products revealed that guava tea had the same strong hemagglutination-inhibition effect as black tea and caused the same marked inhibition of influenza infection as black and green tea. These results indicated that GvEx and commercial guava tea can bind to the surface of influenza virus and inhibit its adhesion to epithelial cells in the respiratory tract thus blocking its infectivity.

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The total daily intakes of three kinds of sugar alcohols (D-sorbitol, D-mannitol, and xylitol) from the foodstuffs purchased in Japan in 1998〜1999 were investigated by a market basket method. One hundred and thirty-two kinds of raw foods and three hundred and forty-four processed foods were collected from markets according to the average intake in Japan, then grouped into seven categories. Each group was homogenized after addition of 80 v/v % ethanol, and D-sorbitol, D-mannitol, and xylitol in prepared samples were determined by HPLC attached with a pulsed amperometric detector (PAD). The total daily intakes of D-sorbitol, D-mannitol, and xylitol were 2350 mg/day, 514 mg/day, and 115 mg/day, respectively. D-sorbitol and D-mannitol were taken from both the processed foods and the raw foods. However, xylitol was taken from only the processed foods.

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A method for simple and rapid determination of imazalil in citrus fruits and banana was examined using solid-phase extraction column (2OH) and high performance liquid chromatography (HPLC). The imazalil was extracted from citrus fruits and banana with n-hexane-ethyacetate (9:1). A 10mL of the extract was passed throush Bond Elut (2OH) cartridge at a flow rate 1mL/min which was previously washed with 5mL of n-hexane-ethyacetate (9:1). The cartridge was washed with 8mL of n-hexane-ethyacetate (7:3) and air-dried under vaccum for 10min. Imazalil in cartridge was eluted with 2mL of HPLC mobile phase. The determination limits of imazalil in citrus fruits and banana were 0.1 μg/g. Recoveries of imazalil from citrus fruits fortifed at the levels of 1.25 μg/g and 2.50 μg/g and banana fortifed at the levels of 0.5 μg/g and 1.0 μg/g were 82.0-91.3%. The mean contents of imazalil in the marketed fruits were 0.57 μg/g in orange (n=19), 0.66 μg/g in lemon (n=18) and 0.74 μg/g in grapefruit (n=28). No imazalil was detected in sweetie (n=5) and banana (n=8).

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The principle of the AW method is that mice sensitized with a protein antigen could exhibit an antigen specific anaphylactic reaction in abdominal wall on challenge with the same antigen. The method is applicable to the detection of protein allergens in food containing unknown components. It has been evident that the appearance of anaphylaxis on abdominal challenge with food samples in the mice sensitized previously with known protein antigen, could indicate the existence of the protein in the food. However, unless the single purified protein antigen as an object of the detection is available, raw food samples themselves have to be used to sensitize the experimental mice. In raw food samples a lot of protein antigens coexist and their concentration varies from food to food. In order to widen the range of application and to ascertain the reliability of the AW method, it is needed to investigate whether the coexisting protein antigens could interact at the both sensitization and elicitation phases, and to determine the dose of antigen required for induction of anaphylactic reaction. To answer these questions, in the present study, we performed three experiments as follows. First we examined the effects of coexisting proteins on the anaphylactic responses by the AW method, using two well-studied protein antigens, hen egg white lysozyme (HEL) and ovalbumin (OVA). Mice sensitized with a mixture of HEL and OVA were challenged with HEL or OVA on the abdominal wall 9 days after sensitization (Table 1). The other mice sensitized with OVA were challenged with a mixture of OVA and HEL 9 days after sensitization. The anaphylactic intensity was estimated by VPV (vascular permeability value) on abdominal wall. VPV of each experimental group was not significantly different from that of the control group. These results suggest that OVA and HEL does not influence each other on both the sensitization and challenge phases. Next, dose-dependent effects of OVA on the sensitization of anaphylactic reaction were studied. Mice were sensitized with various concentrations of OVA (0.0001〜10mg/mL, 50 μL/mouse) and challenged with the fixed concentration of OVA (5 μg/50 μL/site) 9 days later. The observed VPVs were significant at more than 0.01 mg/mL and reached plateau at more than 0.25 mg/mL. Finally we investigated the correlation between induction of anaphylactic reaction in mice and total protein concentration in food. A correlation coefficient between the total protein concentration and the anaphylactic intensity (VPV) was 0.6619. Table 1. Effect of coexisting protein-antigens on the sensitization phase [table]

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Carrageenan is one of a few high-molecular weight substances in nature, which has a lot of sulfur atoms in its molecule. High performance liquid gel permeation chromatography (GPC) can fractionate the high-molecular weight region, and vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometry (ICP-AES) can detect sulfur directly, when GPC/ICP-AES method is used. Unfortunately, it was not acceptable chromatogram on GPC/ICP-AES, which measured the sulfur intensity by ICP-AES, to calculate the average molecular weight of carrageenan, because of higher noise level of the GPC/ICP-AES chromatogram had been obtained. As the results of modifications for GPC conditions by using lambda-type refined carrageenan, we obtained well-fractionated chromatogram on GPC/RI, when the GPC conditions are followed: eluent; 50 mM sodium nitrate, sample content of injected solution; 0.1 mg/mL, elution speed; 0.5 mL/min, elution temperature; 50℃. However, we obtained well-fractionated chromatogram on GPC/RI of lambda-type carrageenan, there was another concern about kappa- and iota-type carrageenan. Kappa- and iota-type carrageenan form a three-fold right-handed double helical structure in aqueous solution. This formation of double helical structure depends on carrageenan concentration, temperature, spices and concentrations of co-cations in aqueous solution. So, it was considered to check if the conditions of ion strength in eluent and elution temperature of GPC was enough to avoid the formation of double helical structure of kappa- and iota-type carrageenan, or not. As the results of optimization of ICP-AES condition and data integration time of GPC/ICP-AES chromatogram, we obtained good chromatogram on GPC/ICP-AES, when the conditions are followed: diffracting order; 4^th, RF-power; 1.50 kW, nebulizer pressure; 200 kPa, PMT voltage; 900 V, data integration time; 4.0 sec. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of lambda-type refined carrageenan we tested, were 446 kDa and 990 kDa respectively, measured by GPC/ICP-AES method in this condition. And the vales of Mn and Mw measured by GPC/ICP-AES method, were almost same as the vales of average molecular weights measured by GPC/RI method respectively. But, when the average molecular weight is calculated via GPC/ICP-AES chromatogram, which measured the sulfur intensity by ICP-AES, the content of sulfur in each molecule must be equal. If the content of sulfur in each molecule is not equal, the sulfur intensity measured by ICP-AES dose not represent the content of solute. Because the average molecular weight of lambda-type refined carrageenan measured by GPC/ICP-AES method was almost same as that measured by GPC/RI, it was considered that the content of sulfur in each molecule of tested carrageenan is almost equal. Regarding this result, it seemed useful to measure the average molecular weight of such carrageenans by GPC/ICP-AES method. The sulfur recoverys of high-molecular weight substances from injected lambda-type refined carrageenan were varied from 97 to 108 % measured by GPC/ICP-AES method. And there was good correlation between lambda-type carrageenan contents in injected solutions and sulfur amounts of high-molecular weight substances measured by GPC/ICP-AES method. This result suggested that it was possible to determine sulfur content of high-molecular weight substances by GPC/ICP-AES method. And it was considered that the GPC/ICP-AES method might determine carrageenan contents in food applications, when it was available which carrageenan was use in a certain food application and the sulfur content came from high-molecular weight substances in its carrageenan was available by GPC/ICP-AES method or total and ionic sulfate analysis.

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Catechins [(-)-epigallocatechin (EGC), (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin 3-O-gallate (EGCG), (-)-epicatechin 3-O-gallate (ECG)] in tea leaf are important natural phenolic materials used in various industries. In this experiment, catechins were easily isolated from water extract of tea leaf by simple procedures as follows: 1) removal of caffeine by the addition of clay which mainly adsorb caffeine and not catechins 2) mass precipitation of catechin- calcium salt by the addition of calcium hydroxide. Catechin- calcium salt obtained as above contained 36.3% of EGCG, 7.3% of EGC, 1.7% of C, 2.0% of EC and 3.9% of ECG (as dry weight) with approximately 10% of calcium. The procedure (preparation of catechin- calcium salt) is especially good for the isolation of EGCG and safety without using of any organic solvents.

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Carrageenan is one of a few high-molecular weight substances, which have a lot of sulfur atoms in their molecules. It may be possible to determine the carrageenan contents from the sulfur contents in high-molecular weight substances by using GPC/ICP-AES method. In this paper, we discussed the determination of carrageenan in the commercial dessert jellies by the method. The recovery of kappa-type refined carrageenan from the dessert jelly containing 0.3% of kappa-type refined carrageenan was 103±10% (n=9). The sulfurs came from the high-molecular weight substances were detected in 9 of 12 commercial dessert jellies we tested, and the sulfur contents in these samples were varied from 0.0447 mg/g to 0.125 mg/g measured by GPC/ICP-AES method. The estimations of carregeenan contents in these jellies were performed as the sulfurs of high-molecular weight substances came from carrageenan only. The values of 7.50 and 20.0 were used as the conversion coefficients derived from the value of sulfate (15-40% on the dried basis) of refined carrageenan and processed eucheuma seaweed listed in Japan's Specifications and Standards for Food Additives Seventh Edition. The results of carrageenan contents in these dessert jellies were ranged from 0.034% to 0.25%. There is the difference of 2.7 times in the conversion coefficients, and usually the other high-molecular weight materials, which have sulfur atoms, are included in food applications. Therefore, there are problems to determine carrageeenan in food applications by using only GPC/ICP-AES method. But, the GPC/ICP-AES method would be helpful to control the conditions of production lines and the qualities of food applications containing carrageenan. Because it was possible to determine the sulfur contents came from the high-molecular weight substances and it was easy to know which materials were added in a certain food application on a food production line.

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Oxytetracycline (OTC) residues in thirty-three meat and fish products were examined, and the effects of cold storage and heating on the levels of residue in pork were investigated. OTC residues were mainly observed in prawns and swine and chicken liver, although every sample was within the tolerance limit. It was found that the level of OTC in pork under refrigeration decreased over time, except for frozen pork dumpling. The OTC levels in pork which OTC had been added were found to be significantly reduced by heating, and the levels were found to decrease more at higher temperature and longer heating time.

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