Structure determination of minor red pigment in carthamus red colorant isolated by preparative LC/MS.

Carthamin is a well-known major pigment in carthamus red colourant. When analysed by HPLC on an ODS column, the colorant separated into two distinct reddish pigments, with both components having almost identical photodiode array spectra. LC/MS analysis suggested one of the compounds was carthamin, whilst the other was an unknown minor pigment. The minor pigment was purified and isolated from the colorant by preparative LC/MS collecting the faction based on monitoring the deprotonated molecule [M-H](-) m/z 953 in electrospray negative-ion mode. The structure was elucidated as a hydroxyethyl ether of carthamin, a novel compound, by means of NMR and HR-FAB-MS analyses.

Isolation and identification of some unknown substances in disposable nitrile-butadiene rubber gloves used for food handling.

In Japan, disposable gloves made from nitrile-butadiene rubber (NBR) are frequently used in contact with foods. In a previous paper, we investigated substances migrating from various gloves made of polyvinyl chloride, polyethylene, natural rubber and NBR. Zinc di-n-butyldithiocarbamate (ZDBC), diethyldithiocarbamate (ZDEC) used as vulcanization accelerators, di(2-ethylhexyl)phthalate (DEHP) used as a plasticizer and many unknown compounds that migrated from NBR gloves into n-heptane were detected by GC/MS. In this paper, six unknown compounds were obtained from one kind of NBR glove by n-hexane extraction and each was isolated by silica gel chromatography. From the results of NMR and mass spectral analysis of the six unknown compounds, their structures are proposed as 1,4-dione-2,5-bis(1,1-dimethylpropyl)cyclohexadiene (1), 2-(1,1-dimethylethyl)-4-(1,1,3,3-tetra methylbutyl)phenol (2), 2,6-bis(1,1-dimethylethyl)-4-(1,1,3,3-tetramethylbutyl)phenol (3), 2,4-bis(1,1,3,3-tetramethylbutyl)phenol (4), 2-(1,1-dimethylethyl)4,6-bis(1,1,3,3-tetramethylbutyl)phenol (5) and 2,4,6-tris(1,1,3,3-tetramethylbutyl)phenol (6). Compound 1 was observed in five of the seven kinds of NBR gloves, and compounds 2-4 and 6, which are not listed in Chemical Abstract (CA), were present in four kinds of gloves.

Molecular weight distribution of carrageenans studied by a combined gel permeation/inductively coupled plasma (GPC/ICP) method.

Degraded carrageenan (known as poligeenan molecular weight: 20 kDa to 30 kDa) causes ulcerative colitis in experimental animals. In this paper, the molecular weight distributions of 29 samples of food-grade refined carrageenans were studied by high performance liquid gel permeation chromatography (GPC) directly connected to vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometry (ICP) (GPC/ICP) as well as GPC/refractive index (RI) detection. All samples of food-grade carrageenan had a major broad peak of high molecular weight which eluted at around 6.5 min in both RI and ICP mode (sulphur and carbon), and each sample of them had no obvious peak of poligeenan (the detection limit was about 5%). The number average molecular weights of these carrageenans ranged from 193 kDa to 324 kDa, and the weight average molecular weights ranged from 453 kDa to 652 kDa based on RI data. Some samples had a few minor peaks which eluted around 10-12 min. These peaks came from ionic sulphate, sucrose or glucose. It was considered that if the data-sampling programme was improved, the GPC/ICP system would become a more powerful technique for evaluation of carrageenan samples containing ionic substances and sugar.

Distribution characteristics of acid-dissolved trace metals of suspended particulate matter (SPM) in Kurashiki, Japan.

This study was undertaken to examine the distribution characteristics of such trace metals in fine particulate (especially with diameters of less than 2.1 microns). The acid-dissolved metals in suspended particulate matter (0.1-9.0 microns) were examined with consideration given to particle size over a 2-year period. It became clear the metals of artificial origin such as Pb, V, Cd, Ni, Cr exist in fine particulates (0.1 or more 2.0 microns), whereas metals of natural origin, such as Ti, Mn, Mg, and Sr primarily exist in coarse particles (2.1-9.0 microns) in Kurashiki City, near the Mizushima Industrial Area in Japan.

[Cause of bisphenol A migration from cans for drinks and assessment of improved cans].

In the previous investigation, we found that some cans for coffee and black tea drinks released large amounts of bisphenol A (BPA) into their contents. Equivalent cans were obtained and the cause of BPA migration was investigated. Equivalent cans A, B and D contained high levels of BPA in the side seam, in the bottom, and in the bottom and the side seam, respectively, while can C contained some level of BPA in the body, which has a large area, therefore, all of them contained high amounts of BPA in their coatings. In the migration test, there was no BPA migration from the cans into water at 60 and 95 degrees C for 30 min, into 20% ethanol at 60 degrees C for 30 min, or into n-heptane at 25 degrees C for 60 min. However, at 120 degrees C for 30 min, equivalent cans released 35-124 ng/mL BPA into the water. The total migration was similar to the total residues of BPA in the can coating and was close to the total amount of BPA in the drinks. Thus, BPA migration from the can coating requires heating to more than 105 degrees C, which is the glass transition temperature of the epoxy resin. Improved cans which contained less than 1/10 as much BPA as the equivalent cans showed very low migration levels, i.e., 3-6 ng/mL.

Isolation and identification of main constituents in an enzymatically hydrolysed licorice extract sweetener.

Licorice extract including glycyrrhizin as the major component is used as a safe sweetener in many countries. Enzymatically hydrolysed licorice extract (EHLE) is also used as a sweetener in Japan. In the present paper, the constituents of EHLE were investigated as a part of the studies for evaluating its quality and safety as a food additive. Three oleanane-type monoglycosides along with glycyrrhizin (1) eand 3-O-[beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]-18beta-liquiritic acid (2) were isolated from EHLE. The structures of the three compounds have been determined to be 3-O-beta-D-glucuronopyranosyl-24-hydroxy-18beta-glycyrrhetinic acid (3), 3-O-beta-D-glucur-onopyranosyl-18beta-glycyrrhetinic acid (4) and 3-O-beta-D-glucuronopyranosyl-18beta-liquiritic acid (5) based on MS and NMR. Compound 4 was the monoglycosylated derivactive of glycyrrhizin (1). Compounds 3 amd 5 are the monoglycosylated derivatives of the minor constituents in licorice extract. They were first isolated from EHLE, and compound 5 was a new compound.

Effects of sialic acid residues of transferrin on the binding with aluminum and iron studied by HPLC/high-resolution ICP-MS.

Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf = 1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf = 1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced.

Identification of principal constituents in enzymatically hydrolyzed coix extract.

The structural elucidation of the main constituents in enzymatically hydrolyzed coix extract, a natural food preservative, was carried out. After peracetylation, five compounds, namely peracetylated forms of glucose, maltose, maltotriose, maltotetraose, and maltopentaose were isolated. The structures were determined by PFG HMQC and HMBC experiments. In addition, by using HPLC with an RI detector, the main components of this coix extract were identified as a mixture of oligosaccharides having one to seven glucose units coupled through alpha-(1-->4) linkages. Since this extract showed no antimicrobial activity, its preservative effect may be caused by its covering of the food surface, thereby blocking contact with air.

[Residual chemicals in silicone rubber products for food contact use].

The residues of additives and other chemicals in 23 kinds of silicone rubber products for food contact use, including nipples, packing and spatulas, were investigated by GC/MS. The packing and spatulas contained 80-480 micrograms/g of BHT, 60-5,830 micrograms/g of di(2-ethylhexyl) phthalate (DEHP), and 60-80 micrograms/g of dibutyl phthalate, while the nipples contained no additives. All of the samples contained 15 to 20 peaks of polydimethylcyclosiloxanes. Dodecamethylcyclohexasiloxane (D6) to tetratriacomethylcycloheptadecasiloxane (D17) were confirmed, and other larger siloxanes up to pentacontamethylcyclopentacosasiloxane (D25) were estimated. A rough estimate of the total cyclosiloxane content was 3,310-14,690 micrograms/g. They might be mainly unreacted materials or by-products, and some of them might be additives. Based on the migration test, no chemicals were released into 20% ethanol at 60 degrees C for 30 min, though DEHP and the polydimethylcyclosiloxanes were released into n-heptane at 25 degrees C for 60 min.

[Migrants from disposable gloves and residual acrylonitrile].

Disposable gloves made from polyvinyl chloride with and without di(2-ethylhexyl) phthalate (PVC-DEHP, PVC-NP), polyethylene (PE), natural rubber (NR) and nitrile-butadiene rubber (NBR) were investigated with respect to evaporation residue, migrated metals, migrants and residual acrylonitrile. The evaporation residue found in n-heptane was 870-1,300 ppm from PVC-DEHP and PVC-NP, which was due to the plasticizers. Most of the PE gloves had low evaporation residue levels and migrants, except for the glove designated as antibacterial, which released copper and zinc into 4% acetic acid. For the NR and NBR gloves, the evaporation residue found in 4% acetic acid was 29-180 ppm. They also released over 10 ppm of calcium and 6 ppm of zinc into 4% acetic acid, and 1.68-8.37 ppm of zinc di-ethyldithiocarbamate and zinc di-n-butyldithiocarbamate used as vulcanization accelerators into n-heptane. The acrylonitrile content was 0.40-0.94 ppm in NBR gloves.

Naringin glycosides alpha-glucosylated on ring B found in the natural food additive, enzymatically modified naringin.

The constituents of the natural food additive, enzymatically modified naringin, were structurally analyzed. Four constituents were isolated from the glucoamylase-treated sample. An NMR analysis revealed that two of them were novel compounds having 4'-O-alpha-glucosyl moieties on ring B of the naringenin aglycone. Both the aglycone and the glucose moiety in naringin are shown to be simultaneously glucosylated.

Analysis of enzymatically glucosylated flavonoids by capillary electrophoresis.

HPCE with UV detection was applied to the analyses of enzymatically glucosylated flavonoids, which are used as natural food additives in Japan. Four items, which have flavonol or flavanone as aglycone, were analyzed. Each of these items is a mixture of glycosides with various lengths of maltooligosaccharide chain. On capillary zone electrophoresis with an untreated fused-silica capillary at alkaline pH, glycosides with longer sugar chains migrated more rapidly. Flavonol glycosides with 1-13 glucose units were distinguished with the borate buffer (pH 10.0). Flavanone glycosides needed higher pH values for good separation than flavonol glycosides.

Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry.

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.

Phytochelatin homologs induced in hairy roots of horseradish.

When exposed to excess heavy metals, plants induce phytochelatins and related peptides (all designated as PCAs). Thus, when hairy roots of horseradish (Armoracia rusticana) were exposed for 3 days to cadmium (1 mM) along with reduced glutathione (2 mM), PCA induction occurred. Moreover, a new family of thiol peptides was detected as well as the previously known PCAs, as revealed by postcolumn-derivatization HPLC. Two were isolated and their structures were identified as (gamma-Glu-Cys)n-Gln (n = 3 and 4) by electrospray ionization-mass spectrometer spectra, this being confirmed by chemical synthesis of the peptides. These new analogs constitute the sixth PCA family identified to date.

Constituents and their sweetness of food additive enzymatically modified licorice extract.

Enzymatically modified licorice extract (EMLE) is a natural sweetener, which is prepared with cyclodextrin glucanotransferase. It is used because of its unique properties such as higher solubility and better taste than those of licorice extract. In the present paper, the structures of six major constituents isolated from EMLE were determined, and their sweetness was studied. The isolated compounds were glycyrrhizin (1), 3-O-[beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]liquiritic acid (2), and their derivatives glucosylated at the C-4 position of the terminal glucuronopyranose with additional one (3 and 4, respectively) and two (5 and 6, respectively) glucose moieties. Compounds 1 and 2 are the major and minor sweet constituents in licorice extract, respectively. Compounds 3-6 are new compounds isolated for the first time. Compound 2 was sweeter than compound 1. Interestingly, compound 3, which is a monoglucosylated derivative of compound 1, was sweeter than compound 4. The sweetness of both compounds was lower than that of the parent compounds, while the lingering sweet aftertaste was markedly improved. Compounds 5 and 6, which have two additional glucose moieties, showed only slight sweetness.

Two new alkaloids from cigarette smoke condensate.

Chemical constituents of MeOH extracts of cigarette smoke were studied. Two new alkaloids, named cigatin A (1), 2-(Pyridine-3-yloxy)-benzene-1',4'-diol and B (2), 2-(4-Methyl-pyridin-3-yloxy)-benzene-1',4'-diol, were isolated from a mainstream condensate of cigarette together with seven known alkaloids. Their structures were determined on the basis of spectral data and chemical methods.

Binding patterns of co-existing aluminium and iron to human serum transferrin studied by HPLC-high resolution ICP-MS.

Serum transferrin (Tf) is an iron-binding glycoprotein. Aluminium in the blood is bound to the transferrin. In the present study, the chemical forms of co-existing Al and Fe bound to human serum Tf were studied by combined on-line HPLC and high-resolution ICP-MS (HPLC-HR-ICP-MS). Samples were subjected to HPLC equipped with an anion-exchange column. The levels of 27Al, 56Fe and 32S, which are interfered with by polyatomic ions such as 13C14N+, 12C15N+ and 12C14N1H+, 40Ar16O+ and 40Ca16O+, and 16O2+, respectively, when using quadrupole ICP-MS, were monitored simultaneously by HR-ICP-MS at a resolution of m/delta m = 3000. Al added to apo-Tf as Al-citrate was preferentially bound to the N-lobe site almost selectively. Al in serum from a healthy person without any in vitro Al spike was present both as AlN-Tf and AlN,FeC-Tf. The chemical states were reproduced in the apo-Tf solution supplemented with Fe (Fe/Tf ratio = 0.6) and Al (Al/Tf ratio = 1) successively. The 32S level was useful for monitoring the protein levels in the HPLC eluate. The clean-up column procedures employed reduced the detection limit for 27Al to 0.1 microgram l-1 (3SB) at the middle resolution.

[Study on the characterizations of genus Curcuma plants on the strains of C. longa L. and C. aromatica Salisb preserved at Izu station of NIHS].

To clarify the characteristics of genus Curcuma plants, we studied the properties of six strains of Curcuma longa L. and two strains of C. aromatica Salisb. preserved at Izu Experimental Station for Medicinal Plants of National Institute of Health Sciences. Six strains of C. longa were classified into three types according to morphological characteristics, rhizome production, and differences in curcuminoid content of rhizome. The curcuminoid content of the rhizomes in each strain ranged from 2.20 mg/g to 55.23 mg/g. Strains showing a high curcuminoid content had a low rhizome yield. No difference was observed between two strains of C. aromatica in terms of morphological characteristics. C. longa can be easily distinguished by differences in the development of tuberous roots and the color of the rhizome cross section.

Direct connection of supercritical fluid extraction and supercritical fluid chromatography as a rapid quantitative method for capsaicinoids in placentas of Capsicum.

The fruits of Capsicum annuum L. are used worldwide as chili peppers and in folk medicines. The pungent components of C. annuum, which are irritants, are called capsaicinoids (CAPS), and the most abundant components are capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. To analyze CAPS in the placentas of Capsicum fruits rapidly and safely, we used a directly connected system of supercritical fluid extraction and supercritical fluid chromatography (SFE/SFC). As a column for SFE/SFC, only a silica-type column was found to be suitable. The CAPS contents in placentas of C. annuum cv. Jalapeno (hot type) and C. annuum cv. Shishitoh (less-hot type) determined by the SFE/SFC method agreed well with those in the range of 0-13.81 mg g(-1) fr. wt determined by the usual extraction-HPLC method. The SFE/SFC method has the advantages of no need for pretreatment and no (or minimal) need for organic solvents. We conclude that this method is useful as a rapid (20 min) and safe screening test for the pungency of various Capsicum fruits.