Inhibition of ATPases by gymnemic acid

A specially prepared and partially fractionated preparation of gymnemic acid compounds were found to be inhibitory to the ATPase system from housefly brain and labellum, and fish brain. Enzyme activity in a homogenate fraction from labellum showed greatest sensitivity to the gymnemic acid preparation. Na⁺-K⁺ ATPase activity from the labellum, which contains the sweet taste sensory receptors, was inhibited by over 92% at a concentration of 4 middot; 10^?6M gymnemic acid. Because gymnemic acid compounds are specific suppressants of sweet receptors, the evidence presented in this paper implies that a portion of the ATPase system may be biochemically important in the sweet-sensing mechanism.     

Taste confusions following gymnemic acid rinse.

The effect of a gymnemic acid (GA) rinse, which simulated a sweet-taste deficit, was measured on human taste perception and identification. Taste ratings showed that GA reduced the intensities of sucrose and aspartame to 14% of pre-rinse levels; over the recovery interval of 30 min, these values increased linearly to 63% of the pre-rinse levels. Repeated presentations of a set of 10 stimuli (five primarily or partly sweet--sucrose, aspartame, and NaCl-sucrose, acid-sucrose and quinine-sucrose mixtures; and five nonsweet--NaCl, KCl, Na glutamate (MSG), quinine HCl and citric acid) for identification following water and GA rinses produced 'taste confusion matrices' (TCMs). Correct identification of the sweet-tasting stimuli was reduced by 23% in presentations closely following the GA rinse, an effect that dissipated with time. Most misidentifications involved sucrose and mixtures containing sucrose. In a second TCM experiment, GA was presented frequently within each session to maintain the sweet taste deficit, which revealed itself as specific confusions. Rinsing with GA impaired discriminability of sweet-nonsweet pairs of stimuli but enhanced discriminability of the aspartame-(NaCl-sucrose) pair. GA had no effect on discriminability of nonsweet stimulus pairs. The results suggest that specific error patterns in the TCM could be used to identify quality-specific taste disorders.     

Study of the effect of gymnemic acid on taste in hamster

Behavioral and electrophysiological experiments with 10 sweetenershave been made to test if gymnemic acid (GA) is able to blockthe response to sweet stimuli in single taste fibres of thechorda tympani proper nerve in hamsters. The hamster was chosenbecause earlier studies show that it is more sensitive to GAthan any other non-human species. Since GA has been shown toaffect the sweetness of many different substances, its effectswere studied on an array of sweeteners. To avoid, however, theinclusion of sweeteners unpalatable to the hamster, the hamsters'liking of this array was tested with two-bottle preference techniquefor –24 h. It was found that acesulfam-K, fructose, glucose,sucrose and xylitol were strongly liked, while the animals showedno preference for aspartame, D-tryptophane, sodium cyclamate,sodium saccharin and thaumatin over water. The summated nerveresponse of these stimuli was then recorded. It was found thatneither thaumatin nor aspartame elicited a response, while theother stimuli gave a good response. Finally, the sweetenerswhich were both preferred in the two-bottle tests and gave anerve response were used as taste stimuli in single fibre experimentstogether with sodium chloride, quinine hydrochloride, citricacid and saccharin. The single fibre recordings were made beforeand after application of 5 mg GA for 3 min on the tongue. Itwas found that GA did not cause any dramatic decrease or disappearanceof the responses to either the sweet or the non-sweet substances.The responses to the sweeteners, however, were more depressedthan those to the non-sweet stimuli.     

Time-dependent inhibition of sucrose sweetness with gymnemic acid: Mode of action

A time-dependent study of the sweet response of sucrose allows a Lineweaver-Burk type of plot (reciprocal of magnitude estimation rate versus reciprocal of sucrose concentration) to be obtained, from which considerable deviation is observed after inhibition of sweetness with gymnemic acid. The plots after inhibition do not appear to be analogous to any of the known forms of competitive, uncompetitive or non-competitive inhibition of enzyme kinetics. Rather a “mixed-type” of inhibition is suggested, combining the attributes of competitive and non-competitive inhibition.     

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.     

Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre

Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl₂ gave maximum response (59.97 mg/gDCW) at 2 mM concentration after a 24 h time period, while, AgNO₃ gave the least response (18.35 mg/gDCW) on incubation of 48 h at 1 mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.     

Molecular Mechanisms for Sweet-suppressing Effect of Gymnemic Acids

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.     

Binding proteins for sweet compounds from gustatory papillae of the cow, pig and rat

The intensely sweet proteins thaumatin and monellin were covalently attached to affinity column supports. Lingual tissue extracts were incubated with the affinity columns which were then eluted with glycine-HCl pH 3.4, the sweet peptide aspartame, or gymnemic acid, which is a sweet taste modifier. SDS-PAGE analysis of eluates from the columns showed that 156 kDa and 47 kDa proteins were the main components from cow fungiform papillae which were specifically bound to thaumatin and monellin. These proteins could be displaced from the column with 0.5 mM aspartame or 0.5 mg/ml gymnemic acid. With circumvallate papillae small amounts of 47 kDa protein were also found. The 47 kDa protein was also the major component bound to a gymnemic acid affinity column and could be displaced from the column with 0.5 mg/ml gymnemic acid. Control experiments with other lingual tissue components indicated that these proteins are localised in the gustatory papillae. Similar protein patterns were also found in extracts of pig fungiform papillae and rat lingual preparations.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

In vitro production of gymnemic acid from cell suspension cultures of Gymnema sylvestre R. Br

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acids. The present work deals with the optimization of a cell suspension culture system of Gymnema sylvestre for the production of biomass and gymnemic acid, which has anti‐diabetic properties. We investigated the effect of inoculum densities (2.5–20.0 g/L), the strength of the Murashige and Skoog (MS) medium (0.25–2.0), carbon source (sucrose, glucose, fructose, maltose), and the concentration of the sucrose (1–8% w/v) to determine their effects on biomass accumulation and production of gymnemic acid. Overall, 10 g/L of inoculum density, full‐strength MS medium supplemented with 2,4‐dichlorophenoxy acetic acid (2.0 mg/L) and Kinetin (0.1 mg/L), and 3% w/v sucrose was found best for the accumulation of biomass and gymnemic acid content (9.95 mg/g dry weight). The results of the current study will be useful for bioprocess and biochemical engineers for large‐scale production of gymnemic acid in cell culture.     

Reduction of saltiness and bitterness after a chlorhexidine rinse

Chronic rinsing with chlorhexidine, an oral-antiseptic, has been shown to decrease the saltiness of NaCl and the bitterness of quinine. The effect of acute chlorhexidine on taste has not been investigated. The purpose of the present study was to examine the effect of acute chlorhexidine rinses on taste intensity and quality of 11 stimuli representing sweet, salt, sour, bitter and savory. All stimuli were first matched for overall intensity so the effects of chlorhexidine would be directly comparable across compounds. As a control treatment, the bitter taste of chlorhexidine digluconate (0.12%) was matched in intensity to quinine HCl, which was found to cross-adapt the bitterness of chlorhexidine. Subjects participated in four experimental conditions: a pre-test, a quinine treatment, a chlorhexidine treatment, and a post-test condition, while rating total taste intensity and taste qualities in separate test sessions. Relative to the quinine treatment, chlorhexidine was found to decrease the salty taste of NaCl, KCl and NH4Cl, and not to significantly affect the tastes of sucrose, monosodium glutamate (MSG), citric acid, HCl and the taste of water. The bitter taste of urea, sucrose octa-acetate and quinine were suppressed after chlorhexidine rinses relative to water rinses, but were only marginally suppressed relative to quinine rinses. Potential mechanisms are discussed.     

REGIONAL VARIATION IN SWEET SUPPRESSION

Differences in the sweet‐blocking efficacy of 2‐(4‐methoxyphenoxy) propanoic acid (PMP) for different sweeteners (sucrose and aspartame) and for various exposure areas of the mouth were found. Twenty participants rated sweetener solutions with and without PMP for sweetness, sourness, saltiness, bitterness and umami for stimulation of anterior tongue, posterior tongue and whole‐mouth areas. For sweetness ratings, suppression was significant for all stimulation areas. In the presence of PMP, stimulation of the posterior tongue yielded significantly higher sweetness ratings than stimulation of the anterior tongue for aspartame but not for sucrose. Sourness and bitterness ratings were significantly higher for anterior tongue than posterior tongue stimulations for aspartame but not for sucrose. The increases in sourness ratings in the presence of PMP were likely because of the sour taste PMP has at the concentration used. Results imply a difference between the front and the back of the tongue in the mechanisms involved in the perception of sweetness.     

Space and time separation of taste mixture components

Under natural conditions, all component qualities of taste mixturesco-exist in space and time on the tongue surface. The significanceof space-time coherence of taste sensations was investigatedby separating mixture components on the tongue. First, it wasshown that a mixture of sucrose plus sodium chloride is judgedto have a qualitatively different sweet taste from a mixtureof sucrose plus quinine sulphate when all taste qualities aretogether in space and time. This occurred even though mixtureswere matched for sweetness intensity, and the saltiness of theformer matched the bitterness of the latter. When, however,mixture components were spatially and temporally separated,the judged quality differences were eliminated. When the componentswere separated in space only, quality difference judgments werereduced in magnitude, but not eliminated. The results are consistentwith the hypothesis that space-time coherence of taste sensationsresult in perceptual blending and a consequent failure of selectiveattention to any single component. It is suggested that tastemixtures can be considered as single tastes even though componentqualities can be reliably identified using other techniques.     

A biphasic model for action of the gymnemic acids and ziziphins on taste receptor cell membranes

A biphasic membrane penetration process for the action of thegymnemic acids and ziziphins on taste receptor cells is proposed.According to this model, the penetrating modifier moleculesinteract first, with the receptor cell plasma membrane surfaceand second, with the lipid interior of the membrane. The initialinteraction is postulated to involve a selective effect on processesfunctional in the transduction and quality specification ofa sweet stimulus and the second interaction to involve a generaldisruption of membrane function and nonselective effect on tasteperception. This biphasic model can account for available chemical,physiological and psyebophysical data, and it makes explicitpredictions which can be tested in future experiments. If themodel holds, it has important implications for the design andinterpretation of psychophysical, neurophysiological and chemicalexperiments that use the gymnemic acids and ziziphins as toolsto study taste perception. Moreover if the model holds, it placesat least one process involved in the specification of a sweetstimulus at the surface of the receptor cell membrane. ^*An earlier version of this paper is included in Kennedy.L.M.,Ph.D. Thesis, Harvard University, 1979 ^**Now at Worcester Foundation for Experimental Biology, Shrewsbury,MA 01545, USA. Reprint requests should be sent to this address.     

Improved gymnemic acid production in the suspension cultures of Gymnema sylvestre through biotic elicitation

Enhancement of secondary metabolite accumulation in cultured plant cells through biotic and abiotic elicitation has been recognised as an important biotechnological strategy. Gymnema sylvestre is a rich source of triterpenoid saponins—gymnemic acids used mainly in the treatment of diabetes I and II. The cell suspension cultures initiated from the leaves and stalks of in vitro-grown plantlets have shown to accumulate large amounts of gymnemic acid. The cell-free extracts of Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis and Escherichia coli were employed as sources of biotic elicitors to study the effect on secondary metabolite production. All the elicitors have shown a positive response in terms of gymnemic acid, with the highest response induced by A. niger [98.65 ± 0.93 mg/gram dry cell weight (gDCW)], 11.2-fold, and the lowest by E. coli (33.25 ± 1.38 mg/gDCW), 3.8-fold, in comparison to the untreated cultures (8.79 ± 0.82 mg/gDCW). The suspension cultures of G. sylvestre can serve as a continuous source of gymnemic acids throughout the year, irrespective of the climatic and geographical barriers.     

Elicitor-enhanced production of gymnemic acid in cell suspension cultures of <Emphasis Type="Italic">Gymnema sylvestre</Emphasis> R. Br.

Cell suspension cultures of Gymnema sylvestre treated with four different elicitors, methyl jasmonate (MJ), yeast extract, chitin and pectin were studied for the production of gymnemic acid as gymnemagenin equivalent, that was analyzed by high performance liquid chromatography (HPLC). All the four tested elicitors induced gymnemic acid production in cell suspension cultures. Highest gymnemic acid content was achieved following treatment with yeast extract (100.47 ± 0.28 mg/l), this was followed by MJ (70.43 ± 0.26 mg/l), pectin (64.19 ± 0.23 mg/l) and chitin (62.72 ± 0.13 mg/l). The addition of elicitors has shown a significant influence on cell growth that affected cell growth compared to respective controls. The highest gymnemic acid production was obtained after 20 days of elicitation in cultures treated with 0.5 g l^−l yeast extract, it was 5.25-folds greater than in control. These results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production. In our present study we could able to overproduce gymnemic acid up to 51.97 ± 0.26 mg l^−l (dry weight basis) in yeast extract treated cell suspension cultures.     

Sweet and sweetness-inducing activities of new triterpene glycosides, strogins

In a previous study we isolated homologues of new oleanane-type triterpene glycosides from leaves of Staurogyne merguensis Kuntze and named them strogins. Strogins themselves have a sweet taste (sweet activity), which diminishes in a few minutes. Subsequent application of cold water to the mouth then elicits a sweet taste (sweetness-inducing activity). In the present study we systematically examined the properties of the sweet and sweetness-inducing activities of strogins. Strogins 1, 2 and 4 had both the sweet and sweetness-inducing activities, while strogins 3 and 5 had no activities. The sweetness- inducing activity in response to cold water lasted for 1 h for strogin 2 and 2 h for strogins 1 and 4. The sweetness-inducing activity was immediately diminished by application of gamma-cyclodextrin to the mouth after strogins were held in the mouth. It seems that the strogins were adsorbed on the gustatory receptor membranes and eliminated by inclusion activity of gamma-cyclodextrin. The structure of strogin resembles that of gymnemic acid, which has antisweet activity. There was competition between strogin 1 and gymnemic acid; treatment of the tongue with strogin 1 before application of Gymnema extract to the mouth reduced the antisweet activity. While the sweetness-inducing activity of curculin in response to water was suppressed by the presence of divalent cations such as Ca2+ or Mg2+, that of strogin was not suppressed by the divalent cations. The changes in the inactive complex between strogin and the sweet receptor site in the adaptation state into the active complex induced by cold stimulation were discussed.      

短时微流水处理对池塘养殖鳙鱼肌肉滋味品质的影响

为探究短时微流水处理对池塘养殖鳙鱼(Aristichthys nobilis)肌肉滋味品质的影响, 采用微流水装置对池塘养殖鳙鱼进行处理, 研究处理时间(0、2d、4d、6d、8d和10d)对池塘养殖鳙鱼肌肉中基本营养成分、游离氨基酸、脂肪酸、核苷酸及其降解产物、甜菜碱含量和感官评分的影响, 以期为提升鳙鱼滋味品质提供新方法。结果表明: 随着微流水处理时间的延长, 鳙鱼肌肉中总游离氨基酸含量、甜味氨基酸含量、腺苷酸(Adenosine monophosphate, AMP)含量和甜菜碱含量呈现先增加后降低的趋势, 均在处理6d时含量最高, 较未处理组分别增加了17.74%、39.45%、28.00%和50.16%; 灰分和鲜味氨基酸含量呈现增加的趋势; 粗脂肪、总脂肪酸含量、饱和脂肪酸含量、单不饱和脂肪酸含量、多不饱和脂肪酸含量、苦味氨基酸含量、酸味氨基酸含量、次黄嘌呤核苷(Hypoxanthine riboside, HxR)和次黄嘌呤(Hypoxanthine, Hx)含量则逐渐降低; 粗蛋白含量在处理过程中无显著变化。滋味分析仪(电子舌)和感官评分结果表明, 微流水处理显著提高了鳙鱼肌肉鲜味和甜味, 降低了其咸味、苦味和腥味, 且处理6d时鳙鱼肌肉的滋味、气味、质地和色泽感官评分最高。故短时微流水处理可显著提高鳙鱼的肌肉滋味品质, 不降低营养品质, 适宜的处理时间为6d。     

Effects of gymnemic acid on sweet taste perception in primates

Application of gymnemic acid (GA) on the tongue depresses thetaste of sucrose in man. This effect, as indicated by electrophysiologicalresponses, has been found to be absent in three nonhuman primatespecies. In the present behavioral study the effect of GA ontaste responses in 22 primate species, with two subspecies,and 12 human subjects has been investigated. In all the nonhumanprimates studied, including the Pongidae which are closely relatedto man, GA did not suppress the response to sucrose, only inman did GA have a depressing effect.     

The effect of cooling the tongue on the perceived intensity of taste

Two experiments were performed (i) to measure the effect ofcooling on the perceived intensity of taste, and (ii) to determinewhether the temperature of the tongue or the temperature ofthe solution was primarily responsible for the changes in perceivedintensity that were observed. The first experiment revealedthat cooling both the tongue and the taste solutions from 36to either 28 or 20°C produced measurable reductions in theperceived intensity of the sweetness of sucrose and the bitternessof caffeine. The saltiness of NaCl and the sourness of citricacid were unaffected by cooling. The second experiment demonstratedthat the temperature of the tongue was the critical factor forproducing the effects on sweetness and bitterness. The latterfinding implies that some of the inconsistencies in the literatureon taste–temperature interactions might have been avoidedif the temperature of the tongue had been routinely controlled.In addition, the importance of lingual temperature suggeststhat thermal effects on taste intensity may often be due tochanges in the sensitivity of the gustatory transduction processrather than to changes in the molecular properties of the tastesolutions.