Search

This note describes a simple and rapid method to separate specific soyasaponins, including the major and multi-functional bioactive species soyasaponin Bb, from the whole soybean flour. The method is based on the difference in solubility of the soyasaponins and other components in methanol, aqueous borax solution, and 1-octanol. First, the whole soybean flour was mixed with methanol to extract hydrophobic components, and the methanol solution was concentrated. Second, the methanol solution was mixed with a larger volume of aqueous borax solution, and the supernatant was acidified to obtain a precipitate. The precipitate was found to be containing mainly four group B soyasaponins. Two of those are non-conjugated molecules, soyasaponins Bb and Bc, and the other two are 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one conjugated ones, soyasaponins βg and βa. All of them have two cis-diol groups in their structure. Therefore, although they are fundamentally insoluble in water, they became soluble in the aqueous medium in the form of divalent anionic species by the borate ester formation, and that they were re-precipitated by the hydrolysis. Furthermore, the non-conjugated group B soyasaponins could be separated by washing with 1-octanol to remove highly hydrophobic soyasaponins βg and βa. The solubility-based technique would be useful as pretreatment in the purification of these group B soyasaponins.

We present a colorimetric method for the detection of aminoglycoside antibiotics such as neomycin (NEO) using a reddish anionic dye, amaranth (AR 3- ). Under acidic conditions, at which NEO exists in fully protonated form (NEOH 6+ ), the AR 3- anion associates with the NEOH 6+ cation to form a precipitate, NEOH(AR) 2 . The precipitate was soluble in a buffer solution of pH 8.5, yielding a reddish solution with an absorption maximum at around 520 nm. Tobramycin and gentamycin, which exist as pentavalent cations under acidic conditions, gave almost the same results. On the other hand, kanamycin, amikacin and streptomycin, which would exist as tri- and tetravalent cations, were not precipitated. Thus, the AR 3- anion could be considered to be an analytical reagent for specific aminoglycosides with polycationic functionality. However, since the precipitation reaction was considerably affected by other anions, a separation method using the tetraphenylborate anion was employed as a pretreatment. The separation method involves precipitating the polycationic aminoglycosides with the tetraphenylborate anion, washing the precipitate with acetonitrile, and re-precipitating the aminoglycosides as hydrochloride salts. Thus, the present method was applied to a microtiter plate assay of the products in an NEO-producing culture broth.

A simple method to separate soyasaponin Bb from a soy extract is presented. This method is based on the difference in the solubility of soyasaponin Bb and Ba and other components into 3:7 and 1:1 (v/v) acetone-water mixed solvents. The crude soyasaponin consisting of soyasaponins Aa, Ab, Ba, and Bb at the 10 wt% level and other components was examined as the soy extract. A 10 mg quantity of the crude soyasaponin was mixed with 1 mL of the 3:7 acetone-water containing 0.1 mol/L HCl, and the supernatant was removed to obtain a precipitate, which was found to contain mainly soyasaponins Bb and Ba. The precipitate was mixed with 0.4 mL of the 1:1 acetone-water containing 0.1 mol/L HCl; the supernatant was transferred, and was mixed with 0.6 mL of water to obtain a precipitate, which was found to contain mainly soyasaponin Bb. The yield was ca. 30%, which may be much higher than that by the conventional preparative chromatographic approach. The separation method is rapid and easy to carry out, and is useful for the preparation of a soyasaponin Bb sample.

Two-dimensional mass spectrometry (MS) analysis of biological samples by means of what is called MS imaging (MSI) is now being used to analyze analyte distribution because it facilitates determination of the existence (what is it?) and localization (where is it?) of biomolecules. Reconstruction of mass image by target signal is given after two-dimensional MS measurements on a sample section. From only one section, we can understand the existence and localization of many molecules without the need of an antibody or fluorescent reagent. In this review, we introduce the analysis of localization of functional constituents and nutrients in herbal medicine products via MSI. The ginsenosides were mainly distributed in the periderm and the tip region of the root of Panax ginseng. The capsaicin was found to be more dominantly localized in the placenta than the pericarp and seed in Capsicum fruits. We expect MSI will be a useful technique for optical quality assurance.

The effects of the cationic polymer ε-poly-L-lysine (εPL) on the enzymatic reaction rate of glucose oxidase (GOD) with ferrocene derivatives having different ionic charges have been investigated by measuring the ferrocene derivative-mediated catalytic current of oxidation by GOD. When negatively charged ferrocenes were used, the bioelectrocatalytic current, which is related to the enzymatic reaction rate, was increased by the addition of εPL. On the other hand, the reaction rates with positively charged ferrocenes were decreased by εPL. These promotion and suppression effects of εPL were remarkable at a certain pH range, where εPL and GOD were charged positively and negatively, respectively. Within this range, the polycationic εPL would be adsorbed onto the GOD surface to enhance the electrostatic interactions of the enzyme with negatively charged substrates, and repulsion with positively charged ones. These findings should be important for practical applications of enzymes.

A simple and convenient method of reducing monosaccharide assay is proposed. A Si(IV)-Mo(VI) solution (pH 4.9) was yellow due to the formation of the 11- and/or 12-molybdosilicate(VI) anions. By the addition of a reducing saccharide, glucosamine, the mixture turned to blue gradually, indicating that the Mo(VI) species was reduced by the saccharide to form a blue molybdosilicate anion. The molybdenum blue formation occurred more quickly when a water-miscible organic solvent, dimethyl sulfoxide, was added to the Si(IV)-Mo(VI) solution. Thus, 0.01% level glucosamine can be determined colorimetrically with microtiter plate. Oligochitosan would not interfere with the determination of the glucosamine at the same concentration. Also, a remarkable blue color development of the Si(VI)-Mo(VI) solution was observed by the addition of glucose. On the other hand, maltose, cellobiose, and water-soluble starch at the same concentration level gave no significant coloration of the reaction mixture. Thus, the present monosaccharide assay can be applied advantageously to evaluate the saccharification to produce glucosamine and glucose.

Two-dimensional (2D) mapping using different chromatographic separations coupled with mass spectrometry is a rapid and simple method for the analysis of a mixture using conventional liquid chromatography mass spectrometry. The 2D map could be created from two different chromatograms obtained with the same detector and different columns or separation methods. In this study, 2D mapping was applied to the analysis of components contained in Panax ginseng, and was evaluated in terms of its effectiveness in the separation of these components. The several glycosides included in Panax ginseng could not be sufficiently separated by one-dimensional chromatography with a reverse phase or a hydrophilic interaction chromatography (HILIC) column, but the components of Panax ginseng could be separated and visualized as a component pattern by 2D mapping. We showed that the components contained in the calli and their quantities were altered by the culture conditions in which the calli were grown by 2D mapping. 2D mapping is expected to be a useful method for visualizing complex component patterns found in glycosides and unknown compounds in foods.

We developed two DNA-conjugated polymers, one based on polyallylamine and the other on polyacrylic acid, for use in DNA chips. A 30-mer single-stranded DNA probe and thioctic acid were covalently attached to polyallylamine as sidechains. The same single-stranded DNA and 3-(pyridyldithio)propionyl hydrazide were covalently attached to polyacrylic acid as sidechains. Both DNA-conjugated polymers could be specifically immobilized onto a gold sensor substrate by a self-assembly technique. The interactions between fully matched DNA and each DNA-conjugated polymer were investigated by surface plasmon resonance. A gold surface modified with either DNA-conjugated polymer recognized fully matched DNA much better than unmatched DNA. The hybridization selectivity and efficiency of DNA-conjugated polyallylamine was optimized by adjusting the pH so as to reduce the effects of cationic polymer sidechains. The hybridization selectivity and efficiency of DNA-conjugated polymers were higher than those of a conventional immobilized thiol-based DNA. The coating of DNA-conjugated polymers reduced nonspecific adsorption of DNA by the gold substrate. DNA-conjugated polyacrylic acid was more selective toward fully matched DNA than was DNA-conjugated polyallylamine. Therefore, DNA-conjugated polymers show promise for application in novel DNA chips.

We developed DNA-conjugated polymer for DNA chip fabrication. A 30 mer probe DNA and disulfide bridges were covalently attached to the polymer side chain. The DNA-conjugated polymer can be specifically adsorbed on a gold substrate surface by a self-assembly technique. The interaction between fully matched DNA and DNA-conjugated polymer was investigated by surface plasmon resonance (SPR) technique. The DNA-conjugated polymer-modified gold surface highly recognized fully matched DNA, rather than unmatched DNA. Therefore, DNA-conjugated polymer can be used for novel DNA chip fabrication.