Toshiaki Imoto

A novel peptide isolated from the leaves of Gymnema sylvestre—I. Characterization and its suppressive effect on the neural responses to sweet taste stimuli in the rat

1. A new substance that suppressed selectively the neural responses of the rat to sweet taste stimuli was isolated from the leaves of Gymnema sylvestre. 2. The substance was proved to be a peptide consisting of 35 amino acids and having a molecular weight of about 4,000. 3. The inhibitory effect on the sweet responses appeared after treating the tongue surface with the peptide at a concentration of more than 1 x 10(-6) M.

Electrophysiological characterization of the inhibitory effect of a novel peptide gurmarin on the sweet taste response in rats

The effect of an anti-sweet peptide, gurmarin purified from the leaves of Gymnema sylvestre, was studied electrophysiologically on taste responses of the rat chorda tympani. The action of gurmarin was highly specific to sweet taste so that responses to various sweeteners including sugars, sweet amino acids and an artificial sweetener, saccharin were all suppressed. The most effective pH at which the rat tongue was treated with gurmarin was found to be 4.5, which corresponds to the isoelectric point of the peptide. At this condition about 5 microM of gurmarin was sufficient to reveal maximal effect and this was still significant at 0.5 microM (2 micrograms/ml). Although the suppressed responses required several hours to attain complete recovery, anti-gurmarin serum shortened the recovery time considerably. On the other hand, intravenous injection of gurmarin did not cause any significant effects on taste responses at all. These results suggest that gurmarin acts on the apical side of the taste cell, possibly by binding to the sweet taste receptor protein.

Three-dimensional structure of gurmarin, a sweet taste-suppressing polypeptide

SummaryThe solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel β-sheet. The average rmsd values between any two structures were 1.65±0.39 Å for the backbone atoms (N, Cα, C) and 2.95±0.27 Å for all heavy atoms. The positions of the three disulfide bridges, which could not be deterermined chemically, were estimated to be Cys3–Cys18, Cys10–Cys23 and Cys17–Cys33 on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in ω-conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in ω-conotoxin.

Inhibitory effect of gymnemic acid on intestinal absorption of oleic acid in rats

Gymnemic acid, a mixture of triterpene glycosides extracted from the leaves of Gymnema sylvestre, is known to inhibit the intestinal absorption of glucose in human and rats. This work examined the effect of gymnemic acid on oleic acid absorption by the method of intestinal perfusion in rats. The results showed the following. (i) Gymnemic acid potently inhibited the absorption of oleic acid in intestine. (ii) This inhibition was dose dependent and reversible. (iii) The extent of inhibition and the recovery progress were extremely similar to that of glucose absorption. (iv) Taurocholate did not affect the inhibitory effect of gymnemic acid on oleic acid absorption, but lowering its concentration facilitated the recovery from the inhibition. (v) The absorption of oleic acid was not affected by other glycosides such as phloridzin, stevioside, and glycyrrhizin. These new findings are important for understanding the roles of gymnemic acid in therapy of diabetes mellitus and obesity.

Enhanced responses of the chorda tympani nerve to nonsugar sweeteners in the diabetic db/db mouse.

Genetically diabetic db/db mice show greater neural and behavioral responses to sugars than lean control mice. The present study examined chorda tympani responses of db/db mice to nonsugar sweeteners and their inhibition by a sweet response inhibitor, gurmarin. The results showed that responses to sucrose, saccharin, glycine,l-alanine, andd-tryptophan, but not tod-phenylalanine, were ∼1.5 times greater in db/db mice than in control mice. Treatment of the tongue with gurmarin suppressed responses to these sweeteners in db/dband control mice, but the extent of suppression was considerably smaller in db/db mice. The magnitudes of gurmarin-sensitive components of the response to sweeteners in db/db mice were not significantly different from those in control mice, whereas the magnitudes of gurmarin-insensitive components in db/db mice were about twice as large as those in control mice. These results suggest that the enhancement of chorda tympani responses in db/db mice to sucrose and other nonsugar sweeteners may occur through gurmarin-insensitive membrane components.Genetically diabetic db/db mice show greater neural and behavioral responses to sugars than lean control mice. The present study examined chorda tympani responses of db/db mice to nonsugar sweeteners and their inhibition by a sweet response inhibitor, gurmarin. The results showed that responses to sucrose, saccharin, glycine, L-alanine, and D-tryptophan, but not to D-phenylalanine, were approximately 1.5 times greater in db/db mice than in control mice. Treatment of the tongue with gurmarin suppressed responses to these sweeteners in db/db and control mice, but the extent of suppression was considerably smaller in db/db mice. The magnitudes of gurmarin-sensitive components of the response to sweeteners in db/db mice were not significantly different from those in control mice, whereas the magnitudes of gurmarin-insensitive components in db/db mice were about twice as large as those in control mice. These results suggest that the enhancement of chorda tympani responses in db/db mice to sucrose and other nonsugar sweeteners may occur through gurmarin-insensitive membrane components.

Molecular mechanisms for sweet-suppressing effect of gymnemic acids

Background: Gymnemic acids inhibit sweet taste responses in humans. Results: Gymnemic acids and glucuronic acid inhibited human sweet receptor T1R2 + T1R3. Conclusion: Interaction between transmembrane domains of human T1R3 and the glucuronosyl group of gymnemic acids is mainly required for the sweet-suppressing effect. Significance: Our model may provide further insights into drug design to modify sensitivity of sweet receptor. Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 μg/ml) inhibited the [Ca2+]i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3.

Enhancement of local anaesthesia action by organic acid salts (I) : possible change of excitability in nerve fibre membrane

Measurements of action potentials recorded from giant nerve fibres of the crayfish abdomen showed that by addition of an organic acid salt to a local anaesthetic solution, the onset of anaesthetic action became more rapid and the duration of action was prolonged. Similar results were also obtained in rat vagal nerves. The chemical structure of compounds having these enhancing effects was found to have, in common, a carboxyl group connected with either a benzene ring or an aliphatic hydrocarbon. Topical application of anaesthetic solutions to the skin of flexor side of the forearm in humans revealed that the duration of anaesthesia evaluated by the apparent decrease of the number of pain points was significantly prolonged by the addition of salicylate. The use of local anaesthetics combined with organic acid salts would be promising in clinical practice to enhance their action greatly.

Interaction of gymnemic acid with cyclodextrins analyzed by isothermal titration calorimetry, NMR and dynamic light scattering

The physiological phenomenon that the antisweet taste effect of gymnemic acid (GA) is diminished by application of γ‐cyclodextrin (γ‐CD) to the mouth was evaluated at the molecular level using isothermal titration calorimetry, NMR and dynamic light scattering. These analyses showed that GA specifically binds to γ‐CD. Thermodynamic analysis using isothermal titration calorimetry revealed that the association constant of GA and γ‐CD is 105−106 m−1 with favorable enthalpy and entropy changes. The heat capacity change was negative and large, despite the change in accessible surface area upon binding being small. These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of γ‐CD. In addition, NMR measurements showed that in solution the spectra of GA are broad and sharpened by the addition of γ‐CD, indicating that unbound GA is in a water‐soluble aggregate that is dispersed when it forms a complex with γ‐CD. Dynamic light scattering showed that the average diameter of unbound GA is > 30 nm and that of GA and γ‐CD complex is 2.2 nm, similar to unbound γ‐CD, supporting the aggregate property of GA and the inclusion complexation of GA by γ‐CD.

High-performance liquid chromatography-atmospheric pressure ionization mass spectrometry of gymnemic acids

Abstract Gymnemic acids (GAs), extracted from the leaves of Gymnema sylvestre, are a mixture of triterpene glucuronides possessing various physiological activities. The molecular masses of GA homologues were determined with high-performance liquid chromatography combined with atmospheric pressure ionization mass spectrometry. A mixture of GAs was chromatographed on an octadecyl silica column eluted with an aqueous methanol solution containing ammonium acetate, and directly introduced into an atmospheric pressure ionization mass spectrometer. Quasimolecular ions of GAs were detected as ammonium adduct ions and/or proton adduct ions. Molecular masses of thirteen different GAs and five compounds not containing glucuronic acid in their molecules were evaluated. Three pairs of geometrical isomers of GAs were found.

Gymnemic acids inhibit rabbit glyceraldehyde-3-phosphate dehydrogenase and induce a smearing of its electrophoretic band and dephosphorylation

Gymnemic acids (GA) inhibited rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity. Binding of GA to GAPDH was observed by surface plasmon resonance measurement. Incubation of GAPDH with GA induced a smearing of the GAPDH band in SDS–PAGE. The GA‐induced smearing was diminished by prior incubation of GA with γ‐cyclodextrin or by GA treatment with NAD. GA treatment did not affect the electrophoretic mobility of glucose‐6‐phosphate isomerase and dehydrogenase. GA treatment diminished the GAPDH band detected by an antibody to phosphoserine, but did not affect the phosphoserine bands of glucose‐6‐phosphate isomerase and dehydrogenase. These results indicated that GA specifically induced dephosphorylation of GAPDH.