[in Japanese]

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Shoot, adventitious root and callus cultures of Ajuga reptans L. were established, and the production of three caffeic acid derivatives [rosmarinic acid (RA), lithospermic acid (LA) and lithospermic acid B (LAB)] in the tissue cultures was investigated. A. reptans plantlets, cultured in vivo and in vitro, accumulated three polyphenols analyzed, although the contents were not so high (RA: 0.001-0.028%, LA: 0.036-0.106% and LAB: 0.002-0.031%, as dry weight). Higher productivity of LA compared to those of RA and LAB, found in A. reptans plantlets, was simiilar to ones which have been also observed in Salvia in Lamiaceae and Lithospermum and Heliotropium in Boraginaceae. Adventitious roots cultured in 1/2 MS liquid medium supplemented with 1 mg/l NAA produced high content (0.045% as dry weight) of LAB, which level was identical with those of the adventitious and hairy root cultures of Ocimum basilicum. The maximum RA content (0.008%, as dry weight) in A. reptans adventitious roots in the same medium was not so high, which level was almost 1/1000 of that of the adventitious and hairy root cultures of Ocimum basilicum and also 1/4 of that of the adventitious root cultures of Heliotropium pervianum. A. reptans calli cultured on 1/2 MS solid medium containing 1 mg/l 2,4-D produced large amount of RA (0.071% as dry weight), while the calli on 1/2 MS solid medium supplemented with 2 mg/l NAA-0.1mg/l BA, produced LA (0.113% as dry weight) as the major polyphenol. The level of the maximum RA content observed in A. reptans calli was almost 1/100 of that of Coleus blumei cells and also 1/10 of those of Lithospermum erythrorhizon and Salvia officinalis cell cultures. In this experiment. A. reptans was clarified to be a resource plant which produce caffeic acid derivatives (RA, LA and LAB), one of the important natural antioxidants. Tissue cultures of this plant, such as shoot, adventitious root and callus, also seemed to be suitable materials for biosynthetic study of these caffeic acid derivatives.

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The composition of three commercial sucrose ester of fatty acid (SE) were analyzed by using TLC and GC. Each commercial SE, DK ESTER^®SS, F-110 and F-10 were consist of mono-ester, mono-, di-, and tri-seter, and di- and tri-seter, respectively. The fatty acids of SE were stearic acid and palmitic acid and the ratio of sucrose/fatty acids of, DK ESTER^®SS, F-110 and F-10 were 1/1, 1/1.7 and 1/2.5, respectively. New method for analysis of sucrose esters of fatty acid in food was established. The foods were extracted with ethylacetate (EtOAc)-tetrahydrofuran (THF) mixture (7:3). The extracts were further purified by using cartridge column (Sep-Pak^® Diol: Waters) to remove the sucrose from foods, completely. The purified SE was hydrolyzed with 5% potassium hydroxide ethanolic solution with reflux for 40 min. Analysis of sucrose in hydrolyzate was performed on high performance ion-exchange chromatography equipped with pulsed amperometric detector (PAD) and refractive index detector (RI). The recoveries of SE from crackers, veinna sausage, and vanilla ice cream were 86.1%〜99.2%, respectively using PAD. The detection limit of sucrose using PAD was 4μg/g.

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The modifying potentials of two madder colors on tumor development were investigated in a medium-term multi-organ carcinogenesis bioassay using male F344 rats. The animals were treated with DEN (100 mg/kg body weight, ip, single injection at the commencement of the study), MNU (20 mg/kg body weight, ip, 4 times during weeks 1 and 2) and DHPN (0.1% in drinking water, during weeks 3 and 4) for multi-organ initiation, and then were given SAN YELLOW AK at dietary levels of 0 (control), 1.5% and 5.0%, SR RED RTM at 2.5% and 5.0%, cochineal (as a negative control) at 5.0%, and S.PB (as a positive control) at 0.05% for 16 weeks. All rats were killed at the end of week 20, and the major organs were carefully examined for preneoplastic and neoplastic lesions. Immunohistochemical demonstration of GST-P positive foci was also employed for quantitative assessment of liver preneoplastic lesion development. No treatment-related changes in clinical signs, body weight, and food consumption were found. Detailed histopathological examination did not demonstrate any enhancement of carcinogenesis with regard to the incidence of preneoplastic and neoplastic lesions in the various target organs/tissues. Quantitative analysis of GST-P positive hepatocytic foci per unit area of liver section did not show any promoting activity on hepatocarcinogenesis. The current results thus revealed that madder colors do not exert tumor promoting or carcinogenic potential in any organ/tissues under the present experimental conditions.

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A survey on the subsidiary colors (Fig. 1), raw materials, and their impurities (Fig. 2) in Food Red No. 2 (amaranth, R2) and Food Red No. 102 (new coccnin, R102) was conducted for the international harmonization regarding the specifications of coal-tar dyes. The analytical results of subsidiary colors in commercial R102 (10 samples of five manufactures) were 0.038〜0.823%, 0.060〜0.324%, 0.008〜0.169%, ND〜0.279%, and 0.07〜0.100%, for Sub A (trisodium salt of 7-hydroxy-8 (6-sulfonaphthyl-2-azo)-1,3-naphthalenedisulfonic acid), Sub B (disodium salt of 4-amino-3-(4-sulfonaphthyl-1-azo)-1-naphthalenesulfonic acid), P6R (pnceau 6R, tetrasodium salt of 7-hydroxy-8-(4-sulfonaphthylazo)-1,3,6-naphthalenetrisulfonic acid), R2, and FRE (fast red E, disodium salt of 6-hydroxy-5-(4-sulfonaphthylazo)-2-naphthalenesulfonic acid), respectively, as showed in Table 1. In one sample (A2), total content of subsidiary colors was 1% which was equal to limit valur of JECFA Specifications. The analytical results of materials and the impurities were 0.005〜0.044%, 0.052〜0.284%, 0.013〜0.196% for TS (trisodium salt of 7 hydroxy-1,3,6-naphthalenetrisulfonic acid), GS (G salt, disodium salt of 7 hydroxy-1,3-naphthalenedisulfonic acid), and NA (naphthionic acid, sodium salt of 4-amino-1-naphthalenesulfonic acid), respectively, and RS (R salt, disodium salt of 3-hydroxy-2,7-naphthalenedisulfonic acid) and SS (Schaeffer's salt, sodium salt of 6-hydroxy-2-naphthalenesulfonic acid) were not detected, as showed in Table 2. All of the samples showed percentages lower than 5%, the maximum limit regulated by JECFA for materials and their impurities. The analytical results of subsidiary colors in commercial R2 (12 samples of two manufactures) were 0.015〜0.773%, 0.219〜1.064%, ND〜0.032%, 0.029〜0.050%, and 0.057〜0.661%, for Sub B, Sub C, P6R, R102, and FRE, respectively as showed in Table 1. THe analytical results of the materials and their impurities wre 0.011〜0.075%, ND〜0.007%, 0.004〜0.072%, 0.052〜0.147%, and ND〜0.011%, for TS, GS, RS, NA, and SS, respectively, as showed in Table 2. All the samples showed percentages that were lower than 3%, which was the maximum limit regulated by JECFA for subsidiary colors, and 0.5%, which was the maximum limit regulated for materials and their impurities. Since the contents of Sub A, Sub B, and Sub C were higher than those of known subsidiary (P6R, FRE, R2 and R102) colors in some cases, it seemed necessary to specify also Sub A, Sub B, and Sub C, which were clarified in the previous report, on evaluating the specifications.

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In order to screen for natural antimicrobial compounds, we carried out halo tests using filter paper disks against ten species of microorganisms at 500mg/ml of extracts obtained from about two hundred plants. Although most extracts were effective against bacteria, the methanol extract of Mosla chinensis Maxim. (Hosoba-yamajiso in Japanese. Labiatae) proved highly inhibitory against not only bacteria but also yeast and fungi (Table 1). Partitioning between H₂O and three successive solvents, hexane, CHCl₃ and EtOAc, and MIC testing against twenty-three species of microorganisms revealed the hexane fraction to exhibit the strongest antimicrobial activity. That is, the MIC values with this fraction were found at the lowest concentration and comparison of the diameter of zone inhibition at the same concentration, demonstrated the hexane fraction to exert the greatest effects against almost all species of microorganisms. Plants of Labiatae, especially such as Thymus, Origanum and Satureja used as spices and food additives, contain essential oils, whose constituents such as thymol, carvacrol and eugenol are known to posess antimicrobial activity. A comparison of antimicrobial activities using the MIC test showed that the methanol extract of M. chinensis was more inhibitory against all soecies of microorganisms than that of Th. vulgaris. In addition, thymol exhibited a wide spectrum of antimicrobial activities, similar to these for M. chinensis (Table 2). Hexane fractions from the leaves, stems, flowers and roots of M. chinensis collected during June to August, were analyzed by GLC in order to determine their volatile constituents, and thymol was detected in all fractions. The content of thymol in leaves, 4.43-13.03% was overwhelmingly higher than in other parts, and maximum levels were observed in leaves collected in August (Table 3). Furthermore, antimicrobial activities of extracts determined by MIC testing were proportional to the tymol content. In conclusion, constituents of the hexane fraction containing essential oils, are the main factors responsible for antimicrobial activity of M. chinensis. The results further suggest the participation of thymol in inhibitory effects against microorganisms.

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Pigments in nine commercially available beet red products and the water-extract from the root of beet (Beta vulgaris LINNE) were analyzed by high-performance liquid chromatography (HPLC). Betanin and isobetanin were the major pigments in the beet red products and the beet root extract, while betanidin and isobetanidin were not detected. Although the content ratio of betanin to isobetanin was 4:1 in the beet root extract, it was approximately 1:1 in all the commercial beet red products. The sum of betanin and isobetanin contents (0.12-0.65%) corresponded to 67-95% of the content of red color in the specifications of JECFA (0.14%-0.70%). The compound that considered as decarboxylated betanin and some yellow degradation products were formed by heat-treatment of the betanin solution. Decarboxylated betanin was also detected in the heat-treated beet red solution and some commercial beet red products.

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We investigated the formation of organochlorine compounds which occurred during the disinfection of cooking tools with sodium hypochlorite. Analysis method added to heat space method for volatile organic compounds such as trihalomethane, and the solvent extract method for middle volatile organic compounds by GC/MS. Usually a sodium hypochlorite solution of around 100-400mg/l was used for disinfection of cooking tools. Chloroform, dichlorobromomethane, dibromochloromethane and bromoform were detected in each concentration of 100, 400, 1000mg/l respectively. Total trihalomethane increased in proportion to the concentration of sodium hypochlorite. And 1,1-dichloroethene, dichloromethane, tetrachloromethane, chloromethane, vinylchlorade were confirmed too. Furthermore the possibility that middle volatile or nonvolatile organoclorine compounds formation is suggested.

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Certain toxic reagents such as benzene, carbon te-tracloride and mercury salts are still used in theofficial analytical methods of food additive stan-dards in Japan. These toxic reagents should be re-placed with safer reagents which be capable of use. Benzene is used in the quantitative analysis of thesodiumdehydroacetate, as a solvent of α-naphtholben-zein in the Japanese standards of food aditives. Acetic acid for nonaqueous titrimetry, glacial and ethanol were tested as a solvent of α-na-phtholbenzein instead of benzene in the quantitativeanalysis, and compared those obtained with benzene. It was found that those reagents were applicable as a solvent inplace of benzene.

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