Reaction of tryptophan with carbohydrates: identification and quantitative determination of novel beta-carboline alkaloids in food

The formation of various carbohydrate-derived beta-carbolines was observed when model reactions of tryptophan with glucose were studied by means of HPLC with diode array detection, as well as by means of HPLC-MS. Isolation of these compounds and subsequent characterization by tandem mass spectrometry and NMR spectroscopy led to the identification of diastereomeric 1-(1,3,4,5-tetrahydroxypent-1-yl)-9H-pyrido[3,4-b]indoles (1a/b), 1-(1,4,5-trihydroxypent-1-yl)-9H-pyrido[3,4-b]indoles (2a/b), and E/Z isomers of 1-(1,5-dihydroxypent-3-en-1-yl)-9H-pyrido[3,4-b]indole (3a/b). HPLC-MS was used to prove the presence of these novel beta-carboline alkaloids in various food samples. In addition, quantitative determination of beta-carbolines 1a, 1b, and 2a/b in numerous products was achieved by means of HPLC with fluorometric detection. Concentrations ranged from 12 to 1922 microg/L for 1a and 1b and from 3 to 644 microg/L for 2a/b. The highest concentrations of all carbohydrate-derived beta-carbolines under study were found in ketchup, soy sauce, and fish sauce.     

Reaction of tryptophan with carbohydrates: mechanistic studies on the formation of carbohydrate-derived beta-carbolines.

Numerous carbohydrate-derived beta-carbolines have been identified for the first time in model reactions of tryptophan with glucose and ribose, as well as in food samples. Extending these structural studies, we performed detailed investigations to elucidate the corresponding intermediates and formation pathway of these alkaloids. Degradation experiments with purified tryptophan glycoconjugates established that only glyco-tetrahydro-beta-carboline-3-carboxylic acids, and not the N-glycosides nor the C-glycoconjugates represented the direct precursors of carbohydrate-derived beta-carbolines. In addition, the significance of the oxidative decarboxylation reaction as the initial step for formation of 1-substituted beta-carbolines was proven. Finally, the stereochemistry of the carbohydrate-derived side chain was studied by means of CD spectroscopy and HPLC-CD experiments. These detailed stereochemical analyses yielded experimental evidence for the racemization steps required for formation of the carbohydrate-derived harman alkaloids and confirmed the proposed reaction pathway.     

Inoculation withGlomus mosseae improves N2 fixation by field-grown soybeans

Summary A field study carried out in a sandy, relatively acid Senegalese soil with a low soluble P content (7 ppm) and low vesicular-arbuscular mycorrhizal (VAM) populations showed that soybean responded toGlomus mosseae inoculation when the soluble P level in the soil had been raised by the addition of 22 kg P ha^–1. In P-fertilized plots, N₂ fixation of soybean, assessed by the A value method, was 109 kg N₂ fixed hat when plants were inoculated withRhizobium alone and it reached 139 kg N₂ fixed ha^–1 when plants were dually inoculated withRhizobium andGlomus mosseae using an alginate bead inoculum. In addition to this N₂ fixation increase (+28%),Glomus mosseae inoculation significantly improved grain yield (+13%) and total N content of grains (+16%). This success was attributed mainly to the low infection potential of the native VAM populations in the experimental site. In treatments without solubleP or with rock phosphate, no effect of VAM inoculation was observed.     

A new approach to enhance growth and nodulation of Acacia mangium through aeroponic culture

This work was designed to determine whether a plant culture method on non-solid media could be used as an alternative for inoculation of Acacia mangium with selected strains of Bradyrhizobium spp. A. mangium seedlings were grown and inoculated with Bradyrhizobium strain Aust13c and strain Tel2 in hydroponics, aeroponics and sand. Aeroponics was found to be the best system of the three, allowing the production of tree saplings 1 m in height after only 4 months in culture. Moreover, compared to plants grown in liquid or sand media, aeroponically grown saplings inoculated with Bradyrhizobium spp. developed a very high number of small nodules distributed all along the root system, resulting in an increase in nitrogen and chlorophyll content in plant tissues. We propose aeroponics as an alternative method to classical soil inoculation procedures for the production of hypernodulated legume tree saplings. Received: 18 July 1996     

Tryptophan-N-glucoside in fruits and fruit juices

In extracts prepared from various fruits as well as in fruit juices a single tryptophan glycoconjugate was detected by HPLC-MS analysis. Product ion spectra demonstrated the N-glycosidic linkage of a hexose moiety to the indole nitrogen. For structure elucidation, the novel tryptophan glycoside was isolated from pear juice and identified as N(1)-(beta-D-glucopyranosyl-(4)C(1))-L-tryptophan by (1)H, HH-COSY and (13)C NMR spectroscopy. Finally, we disclosed the biosynthetic origin of the novel tryptophan metabolite by demonstrating the enzymatic glycosylation of deuterium-labeled tryptophan, which was applied to pear fruit.     

Comparative growth and symbiotic performance of four Acacia mangium provenances from Papua New Guinea in response to the supply of phosphorus at various concentrations

Plant growth performance, the P content in root and nodule tissues, and nodulation and N₂-fixing ability were studied in four provenances of Acacia mangium from Papua New Guinea following different levels of P fertilizer application. A. mangium did not seem to need high levels of P for growth and N₂ fixation. The response by this leguminous tree to the P supply varied significantly according to provenance and to P concentrations in the culture solution. The provenances of A. mangium were classified into three types according to their P response: (1) Growth performance, nodulation, and N₂ fixation of plants were stimulated as concentrations of P increased (provenance PH 482); (2) the maximal effect of P on plant growth was found only at P concentrations higher than 500 M (provenance PH 484); and (3) the plant response to P fertilization was low, even with nutrient solutions containing P concentratins higher than 500 M (provenances PH 483 and PH 485). Provenance PH 483 was distinguished by its low nodulating ability. However, this provenance grew well, probably because of its high N₂ fixation efficiency as expressed by specific acetylene reduction activity and its high P content in nodule tissues. Therefore, in certain cases, these two parameters may be useful criteria in selecting leguminous plants for field use. Statistical analyses of the study results showed that the effect of the factor P supply on N₂ fixation efficiency and nodule development was only significant at P concentrations lower than 250 M whereas the effect of the factor plant provenance was significant regardless of the P concentration used. This observation emphasizes the value of provenance screening in the identification of plants for use in a wide range of soil types.     

Degradation of tetrahydro-beta-carbolines in the presence of nitrite: HPLC-MS analysis of the reaction products.

Motivated by the identification of numerous novel tetrahydro-beta-carboline-carboxylic acids in food samples, we studied the reactions of tetrahydro-beta-carbolines in the presence of nitrosating agents. The anticipated formation of nitroso derivatives from unsubstituted tetrahydro-beta-carbolines, and from tetrahydro-beta-carboline-3-carboxylic acids was indicated by HPLC-MS/MS analysis and validated by the characteristic product ion spectra of the respective nitroso compounds. In addition, oxidative decarboxylation resulted in formation of the corresponding dihydro-beta-carbolines, and in the generation of the beta-carbolines harman or norharman. Subsequently, we studied the reactivity of tetrahydro-beta-carboline-1-carboxylic acids derived from the Pictet-Spengler condensation of indole amines with alpha-oxo acids. Again, in the presence of nitrosating agents the rapid disappearance of the starting material was obvious, but no nitroso derivatives could be observed. Instead, further HPLC-MS/MS studies demonstrated that dihydro-beta-carbolines were the major products of tetrahydro-beta-carboline-1-carboxylic acids. Finally, we demonstrated that freshly isolated nitroso-precursors spontaneously decomposed to yield harman alkaloids. In conclusion, we revealed that nitroso-tetrahydro-beta-carbolines can represent intermediates involved in the generation of beta-carbolines, and we established a novel pathway for the formation of harman alkaloids from nutritional tetrahydro-beta-carbolines.     

Assessment of N2 fixation by Casuarina equisetifolia inoculated with Frankia ORS021001 using 15N methods

Casuarina equisetifolia seedlings, uninoculated or inoculated with Frankia strain ORS021001 were grown for 4.5 months in pouches, then transplanted into 1 m³ concrete containers forming 1 m² microplots. Trees were harvested 6.5 months later when they were 11 months old. N₂ fixation was measured using three methods of assessment: the direct isotopic method, the A-value method and the difference method. Estimations of N₂ fixation during the 6.5 months following transplantation were respectively 3.27, 2.31 and 3.07g N₂ per tree. From these values it was calculated that about 40–60 kg N₂ would be fixed per hectare in a year at normal densities of 10,000 trees ha^?1. The results of this experiment confirm that Frankia strain ORS021001 can be confidently recommended to inoculate casuarinas in the field. Means to improve nodulation and subsequently N₂ fixation by casuarinas are discussed.