The identification and contribution of active components in commercial rosemary extracts as a natural antioxidant using DPPH, which are used as an antioxidative evaluation method, were performed. When two commercial rosemary extracts (manufacture A, powder sample; manufacture B, vegetable oil-liquid sample) were applied to HPLC-DPPH online post-column system, three major peaks (1, 2 and 3) appeared in UV chromatograms at 285nm, and HPLC-separated components corresponding to peaks 2 and 3 reacted with DPPH, resulting that they were detected as major negative peaks in DPPH scavenging chromatograms at 517nm. The results suggested that they would be major antioxidative components in rosemary extracts. The LC-MS/MS and co-injection analyses of authentic samples revealed that compound 1, 2 and 3 were rosmarinic acid (RosA), carnosol (Car) and carnosic acid (CarA), respectively. The contents of RosA, Car and CarA were 4.18, 44.0 and 161mg/g in the rosemary extract A, respectively, and 0.38, 6.30 and 43.3mg/g in the extract B, respectively. The DPPH radical scavenging activity(TEAC, Trolox equivalent activity) of the rosemary extract A, RosA, Car and CarA were 0.350, 2.26, 0.941 and 1.34, respectively, but the activity of rosemary extract B was not measured because the reaction solution was suspended. Calculated from the contents and antioxidant activity, the contribution of Car and CarA to activity of rosemary extract A was estimated as about 75%. On the other hand, the rosemary extract was mixed with 26 antioxidants, and the effects of antioxidants on DPPH radical scavenging activity of the extract were examined by comparison of activity (I_M) of the mixture with the estimated activity (I_E) from individual antioxidants. The ratios of I_M to I_E (I_M/I_E) of the combinations with vanilic acid or p-coumaric acid were almost 0.8, implying that the antioxidants caused antagonistic effects. The ratios of the combinations with caffeic acid or ascorbic acid were more than 1.2, implying synergistic effects.

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A method for the determination of 15 medical components (five erectile dysfunction agents, four hypnotic agents, three appetite suppressants and three oral hypoglycemic agents) in dietary supplements using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed. This method was based on the udenafil (as forbidden component for treatment of erectile dysfunction in Japan) analysis notified by Japanese ministry of health, labour and welfare with some modification. Each medical component was determined with multiple-reaction monitoring (MRM) in both positive and negative modes through electrospray ionization (ESI). Bromovalerylurea, a hypnotic agent, was determined with MRM in negative mode, because this component has no sensitivity in positive mode, while the other 14 medical components were determined in positive mode. Determination of these components was achieved on an ODS column with acetonitrile and aqueous solution (formic acid buffer solution (pH3.5) with 5mmol/L of ammonium formate) as mobile phase with gradient elution method at a flow rate of 0.2mL/min. When the aqueous part of the mobile phase was increased at the beginning of the chromatography compared to the original method, 14 medical components were simultaneously determined in positive mode. Sensitivity of LC/MS/MS is 30-2,500 times higher than results of the other study using HPLC. The recoveries of 15 medical components spiked to a commercially available dietary supplement were 84.7-134.9% and each coefficient of variation was 8.8% or less. It was confirmed that this method is applicable for simultaneous determination of medical components in dietary supplements.

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Quantitative NMR (qNMR) method was applied for the quantification of quercetin in tartary buckwheat (Fagopyrum tataricum L.) noodle. In the reagent market, quercetin is generally provided as quercetin +X hydrate of which the purity is not determined exactly. Hence, if using the reagent as the reference material for LC quantification, the reliability of analysis data will not be assured. qNMR is based on the fact that the signal intensities of a given NMR resonance are directly proportional to the molar amount of that nucleus in the sample, and is able to determine the contents with traceability to International System of Units (SI units). The content of quercetin was calculated from the ratio of the signal intensities of a proton at H-2' on quercetin to eighteen protons of the methyl groups on hexamethyldisilane (HMD) used as the internal standard, after the concentration of HMD was corrected using potassium hydrogen phthalate (PHP), which is one of certified reference material (CRM). In the result, the content of quercetin in tartary buckwheat noodle was determined with SI-traceability to 1.58±0.14mg/g as the anhydrate formula. The quantitative value was verified using general LC method after the purity of reagent was determined exactly. qNMR does not need its reference compound, the calibration curve and separation column like LC method. Our procedure in this study is rapid and simple with overall analysis time of only 10min, and also the result is traceable to SI units.

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近年の研究において、ベニコウジ黄色素の各成分物質が抗ガン作用や抗炎症作用のような生理活性を有することが注目されている。そこで、本研究では高速向流クロマトグラフィー(HSCCC)を用いたベニコウジ黄色素からのキサントモナシンA及びBを効率的に単離精製する手法を開発した。HSCCCの二相溶媒には、酢酸エチル/n-ブタノール/水系(4/1/5,V/V)を用いた。ベニコウジ黄色10mgをHSCCCで測定した結果、キサントモナシンA及びBの分離度は1.8と良好に分離することができた。それらの単離精製された成分を液体クロマトグラフィー/質量分析法(ESI-ポジティブモード)及び紫外可視吸光度法(200-500nm)により、分析評価を行った。その結果、純度95%以上(HPLC)のキサントモナシンA(2.7mg)及びB(0.6mg)を得ることができた。HSCCCで得られたキサントモナシン類は、様々な生理活性試験へ応用できるものと思われる。更に、HSCCCは粗抽出物からの主成分の単離精製に有効な手段と考えられた。

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Dokudami extract, a natural antioxidant agent used as a food additive, is described as a purified ethanol extract of leaves of Houttuynia cordata (Japanese name: Dokudami) and is composed mainly of isoquercitrin, in the Notice (1996) relating to the existing food additives used in Japan. In order to evaluate the quality of the Dokudami extract products, we have analyzed the two products by LC/MS. The results showed that quercitrin and hyperin were more major components than isoquercitrin. However, the Dokudami extract products were confirmed to be made from the leaves of H. cordata, because the compositions of the constituents in the products are similar to those in the extracts of the dried leaves of Houttuynia herba products ('Juuyaku' in Japanese) derived from H. cordata. The antioxidative activities of the food additive products were confirmed by detection of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. These results are also useful for quality evaluation of not only Dokudami extract products as food additives but also various kinds of commercially available Dokudami products as food or folk medicines.

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In our continuing research on guarantee for the safety of dietary supplements derived from medicinal plants, commercial passion flower products were investigated for their botanical origin on the basis of nrDNA ITS1 and cpDNA trnL-F IGS sequences, as well as with analyses of flavone glycoside and β-carboline alkaloid composition using LC-PDA-MS. Both nuclear and chloroplast DNA sequences well distinguished P. incarnata, from other species of the same genus, such as P. edulis, P. caerulea, P. quadranglaris and others. Three ITS1 genotypes were found in passion flower products which were assigned to P. incarnata, P. edulis and P. edulis f. flavicarpa with reference to the sequences of referential Passiflora plants. Flavone glycoside composition showed the species- and forma-specific variation and the profile of each product supported the results of DNA sequence analyses. Fourteen passion flower products were analyzed for their source plant species, and were shown to be made from P. incarnata (nine samples) and P. edulis sensu lato (five samples) on the basis of the DNA and LC-PDA-MS analyses. Furthermore, β-carboline alkaloids such as harmine and harmaline which were reported as the constituents of P. incarnata were not detected in the products. Plant materials legally restricted to medicinal use in Japan are specified by their scientific names and listed on the Pharmaceutical Affairs Bureau Notification. However, those for general herbal products are not specified. According to the results, plant material used for general herbal products are suggested to be specified by scientific names in order to ensure their safty.

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This study compared the changes of physical properties between normal and burnt ordinary muscle of cultured yellowtail (Seriola quinqueradiata). Anti-α-actinin IgG was prepared from rat serum immunized with purified α-actinin in the ordinary muscle of cultured yellowtail. The α-actinin in burnt muscle was examined by SDS-PAGE, immunoblotting and immunocytochemistry. The burnt muscle showed the color L^* tended to rise rapidly, the expressible water sustained higher and the breaking strength decreased gradually during storage, compared with the normal samples. It was found that the limiting digestion of α-actinin in burnt muscle of cultured yellowtail started just after slaughter and native α-actinin was detected at on 4hour storage. According to immunocytochemical method, antigenic activity of α-actinin was detected on Z band of ordinary muscle cells and non-specific reactions were not detected on another organelles. Antigenic activity of α-actinin in burnt muscle was diffused from Z band to long axis of myofibril just after slaughter and it decreased during storage. These results suggested that limiting digestion of α-actinin influences deterioration of burnt muscle of cultured yellowtail.

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In recent years, the number of patients with allergies to crustaceans such as shrimp and crab has increased in Japan. According to a 2008 epidemiological survey of food allergies in Japan, shrimp is the sixth most frequent cause of food allergies following the major allergenic ingredients such as egg, milk and wheat. Cross-reactivity in allergy symptoms has been speculated between shrimp and crab since approximately 65% of patients with shrimp allergy have also been reported to respond to crab allergens, although the frequency of crab allergy is less than that of shrimp allergy. In June 2008, the Ministry of Health, Labor and Welfare in Japan enforced a mandatory food labeling system of shrimp and crab in processed foods. Therefore, we surveyed the contents of soluble proteins and DNA of crustaceans in 77 seafood products (29 ground and 48 processed fish pastes) using ELISA and PCR, respectively. Crustacean proteins were detected in 75.9% (M kit) and 79.3% (N kit) of ground fish pastes. With shrimp and crab PCR, 62.1% and 3.4% of the samples, respectively, were judged to be positive. In addition, in 56.3% (M kit) and 72.9% (N kit) of processed fish pastes, crustacean proteins were detected, and the percentage of positive samples were 72.9% and 2.1% in each PCR. Consequently, the ground and processed fish paste products all contain crustaceans at high frequency, probably due to unintentional natural contamination. A high concentration of crustacean soluble protein was detected especially in the ground fish pastes made from small size fishes.

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Because of the use of mouthwash and liquid toothpaste, the intake of benzoic acid, sodium benzoate, and parabens combined as preservatives can be a concern. Therefore, we investigated the conditions of use for 50 men and women between the ages of 20-60 years. Next, an investigation of the amount of intake in commercially available products containing benzoic acid and methylparaben was performed. Participants using mouthwash and liquid toothpaste were 18 out of 50 (36%), 12 out of 18 used them once a day, which is the most common frequency (67%), and the average use was 1.4 times. Moreover, the amount used each time for most of the participants was within a suitable amount ranging from 10 to 20ml. The result of examining the intake of 15 adult men and women showed that the estimated intake of benzoic acid was 4.6mg/day for mouthwash and 5.0mg/day for liquid toothpaste. Furthermore, the estimated intake for methylparaben was 4.1mg/day and 1.0mg/day, respectively. Although benzoic acid, sodium benzoate, and parabens are components used as preservatives for food additives, it has been revealed that their intake because of the use of mouthwash and liquid toothpaste was more than the intake with food. However, a comparison with the ADI levels showed that it is not at a level that would immediately influence a person's health.

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日常生食用として摂取される植物性食品(果実類、野菜類、豆類)のうち、24科46品目の組織外果皮および果肉内部の細胞内キチナーゼ活性分布を調べた。組織生重量あたりキチナーゼ活性が高い品目は、カキノキ科カキノキ果皮・果肉、ウリ科ハーデイスメロン果肉、マスクメロン果肉、ウルシ科マンゴー果皮・果肉、クスノキ科アボカド果皮、アケビ科ムベ果実およびマタタビ科キウイフルーツ果肉であった。最もキチナーゼ活性が高かったカキノキ科に絞り、そのうち品種を特定した5品目果皮について検討した結果、富士柿(愛媛県産)は、果皮生重量100gあたり1.080U及び果皮タンパク質含量あたり68.2U/mg proteinの酵素活性を示した。通常購入できる食用農作物として入手可能な植物組織に高いキチナーゼ活性が分布していることから、これらの作物を生物農薬の基材として活用する可能性を提示しているものと考えられる。

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市販ローヤルゼリー(RJ)含有健康食品中のトランス型10-ハイドロキシ-2-デセン酸(10-HDA)の高速液体クロマトグラフィーを用いた簡易で迅速な定量法を開発した。試験方法は、RJ中の10-HDAをメタノールで抽出し、遠心分離後の上澄液をHPLCに導入した。HPLC分析は、C18カラムを使用し、移動相として0.6%リン酸-メタノール(1:1)を0.8mL/minで流し、215nmで検出した。10-HDAの検量線は、1-100μg/mLの濃度範囲で直線性を示した(r>0.999)。試料として、3種(錠剤、液剤、未加工RJ)を用いて添加回収率を求めたところ、93.0-110.4%と良好な結果が得られた。本分析法を市販の21製品に適用したところ、製品中の10-HDA濃度は0.2-45.8mg/gであった。さらに10-HDA測定値を基に、製品のRJ含量を算出したところ、製品の表示値に近い値であった(58.1-94.6%)。

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