Isolation of glucosinolates and the identification of o-(α-l-rhamnopyranosyloxy)benzylglucosinolate from Reseda odorata

A method has been developed for the quantitative isolation of glucosinolates by ion-exchange chromatography and high voltage electrophoresis avoiding strongly alkaline and acidic conditions. The compounds were identified by ¹H and ¹³C NMR spectroscopy and through the products arising from enzymatic, acid and alkaline hydrolysis. The method is well suited for the isolation and identification of glucosinolates containing aglucone parts which produce non-volatile compounds on enzymatic hydrolysis. The method has been used in the isolation and identification of 2-hydroxy-2-methylpropylglucosinolate from Reseda alba, 2-hydroxy-2-phenylethylglucosinolate from R. luteola and a new glucosinolate, o-(α-l-rhamnopyranosyloxy)benzylglucosinolate, occurring in R. odorata. The glucosinolate content in different parts of this plant has been determined and the metabolism of glucosinolates is briefly discussed.     

Esterified long-chain hydroxy acids in green and senescent parts of the peat moss Sphagnum fuscum

The bifunctional long-chain acids in extracts and alkaline hydrolysates of extractive-free tissues from the green and senescent parts of Sphagnum fuscum were investigated by GC and GC/MS. A series of C₁₄– C₂₆hydroxy acids was liberated by alkaline hydrolysis of senescent tissue demonstrating the presence of insoluble polymeric lipid esters in the moss. In the corresponding hydrolysates of the topmost green parts of the shoot the amounts of these acids were significantly smaller. It is suggested that the presence of such polymeric esters in S. fuscum contributes to the cell-wall resistance of this peat-forming moss.     

Glucosinolates in Sesamoides canescens and S. pygmaea: Identification of 2-(α-l-arabinopyranosyloxy)-2-phenylethylglucosinolate

A new natural product, 2-(α-l-arabinopyranosyloxy)-2-phenylethylglucosinolate, has been isolated from Sesamoides canescens. This glucosinolate together with 2-hydroxy-2-phenylethylglucosinolate and 2-phenetliylglucosinolate in a 1:1:1 ratio constitutes about 90 % of the total glucosinolate pool in green parts of the plant. Phenethylglucosinolate constitutes about 70 % of the total glucosinolate pool in green parts of S. pygmaea together with minor amounts of the two other glucosinolates. In addition, both plants contain at least seven other glucosinolates. The structure of the new natural product has been confirmed by transformations into d-glucose, l-arabinose, N-(2-(α-l-arabinopyranosyloxy)-2-phenylethyl)thiourea, 3-(α-l-arabinopyranosyloxy)-3-phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid, respectively. The significance of this investigation is briefly discussed in relation to the methods used in glucosinolate analysis, chemotaxonomy and possible catabolic transformation of glucosinolates into amines.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide by thermolysin

The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the K_m for the enzyme substrate interaction is 2.5 × 10^?3m at pH 7.2. This K_m is an order of magnitude less than what has been previously assumed to be the K_m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10^?3m substrate and is based on the assumption that the substrate concentration is much less than the K_m. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10^?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K_m.     

Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

(1) The hydrolysis of ³²P- or myo-[2-³H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca²⁺ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca²⁺ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[³H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.     

Synthesis and in vivo testing of a bromobutyl substituted 1,2-dithia-5,9-diazacycloundecane: a versatile precursor for new 99mTc-bis(aminoethanethiol) complexes

7-(4′-Bromobutyl)-3,3,11,11-tetramethyl-1,2-dithia-5,9-diazacycloundecane (6) an intermediate for the preparation of new ^99mTc-bis(aminoethanethiol) complexes (^99mTc-BAT) was synthesized from the corresponding 7-(4′-phenoxybutyl) derivative (5) by ether cleavage with HBr/AcOH. To demonstrate its versatility as an alkylating agent, 6 was reacted with the amines piperidine, morpholine, NH₃ and l-phenyl-1,3,8-triazaspiro(4,5)decan-4-one, yielding the corresponding N-alkylated amines. Mild ring opening affording the BAT-ligands was achieved by reductive cleavage of the disulphide bonds with threo-2,3-dihydroxy-1,4-dimercaptobutane. The ^99mTc-BAT complexes prepared by the tin-reduction method proved to be stable under in vitro conditions. With the exception of the 7-(4′-aminobutyl) substituted one, the ^99mTc-BAT complexes revealed octanol-buffer partition coefficients (P) of log P > 1 at physiological pH. The complexes proved to be neutral and the amount of ultrafiltrable ^99mTc-BAT complex varied between 8–18%. In contrast to the good in vitro characteristics, the brain uptake values in CD-1 mice were comparably low.     

Effect of zinc on the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell)

The effect of Zn²⁺ ions (in the form of ZnCl₂) in the ceoncentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell). In the “long-term” experiment zine ions influenced the seedlings during eight days of germination, whereas in the “short-term” experiment zinc ions acted only 72 h on seven days old intact seedlings. The biosynthesis of indole glucosinolates has been followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into both glucosinolates in experiments with, hypocotyl segments of the rape seedlings. Zinc ions at chronic “long-term” application increased the glucobrassicin and neoglucobrassicin level in the seedlings. The neoglucobrassicin content especially was increased. A “short-term” application of zinc ions increased the level of both glucosinolates at higher and lower concentrations, whereas medium concentrations (10^?4 and 10^?5 M) lowered their level. Zn²⁺ ions lowered absorption of³⁵SO₄ ^?2 ions by hypocotyl segments and simultaneously lowered the incorporation of³⁵S into glucobrassicin. On the contrary, the incorporation of³⁵S into neoglucobrassicin and proteins was stimulated. Zinc ions do exhibit a specific effect on neoglucobrassicin biosynthesis, on membrane permeability as against sulphate ions and on the incorporation of sulphur into proteins.     

ω-Methylthioalkylglucosinolates and some oxidized congeners in seeds of Erysimum rhaeticum

The glucosinolates in seeds of Erysimum rhaeticum Schleich. ex DC. have been identified by structure analysis of their sulfur-containing enzymic hydrolysis products, comprising 5-methylthiopentyl and 6-methylthiohexyl isothiocyanate, the corresponding 3-hydroxylated isothiocyanates, and the sulfoxides and sulfones of the latter. The phytochemical results are evaluated in terms of their possible taxonomic significance.     

Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

The location and mobilization of polyphosphates in response to an amine-induced alkaline stress were studied in the halotolerant alga Dunaliella salina. The following observations suggest that polyphosphates accumulate in acidic vacuoles: (a) Accumulation of large amounts of polyphosphates is manifested as intravacuolar dense osmiophilic bodies in electron micrographs. (b) Uptake of amines into the vacuoles induces massive hydrolysis of polyphosphates, demonstrated by in vivo ³¹P-nuclear magnetic resonance, and by analysis of hydrolytic products on thin layer chromatograms. The analysis indicates that: (a) Polyphosphate hydrolysis is kinetically correlated with amine accumulation and with the recovery of cytoplasmic pH. (b) The major hydrolytic product is tripolyphosphate. (c) The peak position of the tripolyphosphate terminal phosphate in nuclear magnetic resonance spectra is progressively shifted as the cells recover, indicating that the pH inside the vacuoles increases while the pH in the cytoplasm decreases. (d) In lysed cell preparations, in which vacuoles become exposed to the external pH, mild alkalinization in the absence of amines induces polyphosphate hydrolysis to tripolyphosphates. It is suggested that amine accumulation within vacuoles activates a specific phosphatase, which hydrolyzes long-chain polyphosphates to tripolyphosphates. The hydrolysis increases the capacity of the vacuoles to sequester amines from the cytoplasm probably by releasing protons required to buffer the amine, and leads to recovery of cytoplasmic pH. Thus, polyphosphate hydrolysis provides a high-capacity buffering system that sustains amine compartmentation into vacuoles and protects cytoplasmic pH.     

Glucosinolate contents in maca (Lepidium peruvianum Chacón) seeds, sprouts, mature plants and several derived commercial products

Several products derived from processed maca hypocotyls (Lepidium peruvianum Chacón, previously known asL. meyenii Walp.) were surveyed for glucosinolate content and quantified by HPLC analysis. These included pills, capsules, flour, liquor, tonic and mayonnaise. Different plant organs such as fresh hypocotyls and leaves, seeds, dry hypocotyls, and sprouts were also included in the survey. The most abundant glucosinolates detected in fresh and dry hypocotyls and leaves were the aromatic glucosinolates, benzylglucosinolate (glucotropaeolin) and p-methoxybenzylglucosinolate. Maca seeds and sprouts differed in profile from hypocotyls and leaves due to the modification of benzylglucosinolate. No glucosinolates were detected in liquor and tonic, while mayonnaise had only trace amounts of those glucosinolates. It had instead allylglucosinolate (sinigrin), which is an aliphatic glucosinolate. The pills, capsules and flour had the same glucosinolates as those observed in hypocotyls, but in variable amounts. The richest sources of glucosinolates were seeds, fresh hypocotyls and sprouts, in that order.     

Diamine Oxidase from Cultured Roots of Hyoscyamus niger: Its Function in Tropane Alkaloid Biosynthesis

Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective K_m values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants V_max/K_m for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.     

Seed glucosinolates of fourteen wild Brassica species

Seed glucosinolates have been determined for 14 wild Brassica species, by micro-scale GC analysis of silylated derivatives. Of these, 12 were investigated for the first time. The majority of taxa exhibited high alkenylglucosinolate levels, although prop-2-enylglucosinolate appears to be generally absent. Other known methionine-derived glucosinolates predominate in B. tournefortii, B. elongata and B. deflexa. Phenylalanine-derived 4-hydroxybenzylglucosinolate is characteristic of section Brassicaria plants and represents the first finding of this glucosinolate in authenticated Brassica material.     

Effect of copper on the content and the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated rape seedlings (Brassica napus var.arvensis (Lam.) Thell.)

The influence of Cu²⁺ ions (in the form of CuCl₂) in the concentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell.). Ions Cu²⁺ acted on the seedlings either chronically from the beginning of the germination or acutely, during 3 to 72 h, on seven days old seedlings. The biosynthesis of both glucosinolates was followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into them in hypocotyl segments from seven days old intact etiolated seedlings. After the entry of small amounts of Cu²⁺ ions into the plants, stimulation of the glucosinolates formation occurs, as was found after three h action of Cu²⁺ ions. After the entry of a greater amount of Cu²⁺ ions into the plant, harmful effects appear, as was found after chronic two days action or after 24 and 48 hours acute action of Cu²⁺ ions. Later further stimulation of glucosinolate formation occurs, probably due to enhanced metabolism during reparation processes, as was manifested after chronic action of Cu²⁺ ions lasting four and eight days. The optimal effect of copper was found mainly in the concentration range 5×10^?4 M to 10^?5 M. Ions Cu²⁺ in higher concentration increased the uptake of sulphate ions by hypocotyl segments, and in lower concentrations increased the incorporation of³⁵S from³⁵SO₄ ^2? into the proteins.     

Studies of the Biochemical Background to Differences in Glucosinolate Content in Brassica napus L. II. Administration of Some Sulphur-35 and Carbon-14 Compounds and Localization of Metabolic Blocks

The aim of the investigation was to study the differences in the metabolism of substances that are utilized in the synthesis of glucosinolates between the Brassica napus cv. Bronowski, which is very low in glucosinolate content, and a cultivar (cv. Regina II) that contains approximately average amounts of these compounds. By experiments in which the plants were grown in nutrient solutions supplied with sulphate-³⁵S, it was shown that the rate of sulphate uptake was similar in the two cultivars. No accumulation of intermediate metabolites could be demonstrated by autoradiography in Bronowski. Sulphate-³⁵S, methionine-³⁵S, methionine-2-¹⁴C, 2-amino-6-(methylthio)caproic acid-2-¹⁴C, and S-(β-d -glucopyranosyl)-4-pentenethiohydroximic acid (desulpho-3-butenylglucosinolate) (glucose-U-¹⁴C or ³⁵S) were fed to shoots of the two cultivars. After incubation, the plant material was extracted with methanol. The extracts were separated into various fractions, and in some experiments glucosinolates or derivatives of their degradation products were isolated. When measuring the radioactivity of the various fractions or isolated products, the incorporation of radioactivity into glucosinolates was found to be poor in Bronowski from sulphate, methionine, and 2-amino-6-(methylthio)caproic acid. Desulpho-3-butenyl-glucosinolate was an efficient precursor of 3-butenylglucosinolate in Bronowski, but a poor precursor of 2-hydroxy-3-bute-nylglucosinolate, which suggests a metabolic block at the hydroxylation step in this cultivar. In Regina II desulpho-3-butenylglucosinolate was a good precursor of both 3-butenylglucosinolate and of 2-hydroxy-3-butenylglucosinolate, which demonstrates that these glucosinolates may be synthesized without prior formation of the corresponding co-methylthioalkyl glucosinolates and that the hydroxylation can take place after the formation of desulpho-3-butenylglucosinolate. The results indicate that the low glucosinolate content of Bronowski is caused by block(s) in the separate pathway leading to the biosynthesis of glucosinolates.     

1H, 13C and 199Hg NMR characteristics of new amine-mercuric chloride complexes

Reduction of some N-alkylimines has been achieved with NaBH₄ to give the corresponding secondary amines with high yields (85–95%). These amines were characterized on the bases of their ¹H and ¹³C NMR spectra. The reaction of these amines with mercuric chloride to afford the corresponding complexes was found to occur through a weak dative bond between the nitrogen lone pair of electrons and the mercury atom to form HgCl₂L₂ complexes. The ¹H, ¹³C and ¹⁹⁹Hg NMR chemical shifts have been obtained as well as ¹J(¹³CH) and ²J(¹³CH) coupling constants. Labelling with nitrogen-15 revealed that there is a weak coupling between the nitrogen and the ¹⁹⁹Hg.     

A chromatographic technique for the analysis of oxidized metabolites: Application to carcinogenic N-hydroxyarylamines in urine

A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe⁺³-bathophenanthroline colorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, 1- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The N-glucuronide has been shown to undergo acidic hydrolysis in the urine to release the free N-hydroxyarylamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the 1-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2-naphthylamine in a variety of species.     

Glucosinolates in <Emphasis Type="Italic">Brassica</Emphasis> foods: bioavailability in food and significance for human health

Glucosinolates are sulphur compounds that are prevalent in Brassica genus. This includes crops cultivated as vegetables, spices and sources of oil. Since 1970s glucosinolates and their breakdown products, have been widely studied by their beneficial and prejudicial biological effects on human and animal nutrition. They have also been found to be partly responsible for the characteristic flavor of Brassica vegetables. In recent years, considerable attention has been paid to cancer prevention by means of natural products. The cancer-protective properties of Brassica intake are mediated through glucosinolates. Isothyocianate and indole products formed from glucosinolates may regulate cancer cell development by regulating target enzymes, controlling apoptosis and blocking the cell cycle. Nevertheless, variation in content of both glucosinolates and their bioactive hydrolysis products depends on both genetics and the environment, including crop management practices, harvest and storage, processing and meal preparation. Here, we review the significance of glucosinolates as source of bioactive isothiocyanates for human nutrition and health and the influence of environmental conditions and processing mechanisms on the content of glucosinolate concentration in Brassica vegetables. Currently, this area is only partially understood. Further research is needed to understand the mechanisms by which the environment and processing affect glucosinolates content of Brassica vegetables. This will allow us to know the genetic control of these variables, what will result in the development of high quality Brassica products with a health-promoting activity.     

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis–Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts.     

Preparation and characterization of a new trans-platinum glycocholate complex

The synthesis and characterization of a new water-soluble cytotoxic transplatinum compound are described. The new compounds, trans-[Pt^II(GC)₂(NH₃)₂], is a transplatinum derivative containing an N₂O₂ donor set, using glycocholate (GC, a bile acid) ligands. The new compound has been characterized by infrared and NMR spectroscopies, as well as mass spectrometry. The FTIR spectra show a separation between νCOOas and νCOOs stretching bands in agreement with a monodentate binding mode of carboxylate group attached to platinum. The new compound is compared with cis-[Pt^IICl(GC)(NH₃)₂] in their cytotoxic properties against three cell lines (HELA229, A2780 and A2780cisR). Both compounds showed a poor inhibition on the growth of these cell lines in comparison with cis-platin (cis-DDP). Bile acids are involved in the enterohepatic cycle. This cycle can be useful for directing vectorially certain drugs for the treatment of diseases of the organs linked to this cycle. The next step of cytotoxicity assays will be performed in our laboratory, using cell lines related to these organs.