Synthesis of ellagic acid O-alkyl derivatives and isolation of ellagic acid as a tetrahexanoyl derivative from Fragaria ananassa.

Ellagic acid [1] is a gallic acid dimer that occurs in plants, fruits, and nuts, either in its free form, or in a series of ellagitannins, or as a glucoside. It has been shown to inhibit cancer induced by several types of chemical carcinogens including polycyclic aromatic hydrocarbons, N-nitrosamines, aflatoxin, and aromatic amines. It has been extracted from a number of fruits, including strawberries; however, its presence in the extracts was determined only by hplc connected with a diode array detector. In the present report, ellagic acid was isolated as a tetrahexanoyl derivative 2 from Fragaria ananassa and identified by 13C and 1H nmr and ms. The 13C-nmr shifts of the aromatic carbons adjacent to a hexanoyloxy group were assigned using two new synthetic model compounds: 3,3'-dihexanoyloxydiphenic-2,2',6,6'-dilactone [3] and 4,4'-dihexanoyloxydiphenic-2,2',6,6'-dilactone [4]. Two new derivatives of ellagic acid [1],3,3'-di-beta-D-glucopyranosylellagic acid decaacetate [5] and 3,3'-di-n-octyl-4,4'-dihexanoylellagic acid [7], were also synthesized. Both derivatives were less effective as inhibitors of benzo[a]pyrene tumorigenesis in the lungs of strain A/J mice than ellagic acid.

Ellagic acid protects rat embryos in culture from the embryotoxic effects of N-methyl-N-nitrosourea.

Ellagic acid is a naturally occurring plant phenol that has demonstrated anticarcinogenic and antimutagenic activity in several test systems. Given the common proposed etiopathogenic processes of mutagenesis, carcinogenesis, and teratogenesis induced by genotoxic chemicals, the present study was initiated to determine whether ellagic acid would protect rat embryos in culture from the teratogenic effects of N-methyl-N-nitrosourea (MNU). Ellagic acid alone (as used in these experiments; 50 microM in DMSO) was not embryotoxic. Ellagic acid (50 microM) significantly (P less than 0.01) prevented MNU (75 microM)-induced effects including mortality (absence of heart beat), abnormal formation of the cephalic neural tube derivatives, and delayed differentiation as assessed by a morphological scoring system. These embryoprotective effects were dose responsive. Sequential treatment of embryos with ellagic acid followed by MNU in fresh media also was embryoprotective with no diminution of effect. The site at which ellagic acid interrupts the critical teratogenic events induced by MNU is apparently within the embryo and/or placenta. This model of chemical embryoprotection may be useful in determining the role of cell death and/or mutation in the teratogenic mechanism of action of methylating agents.

Inhibitory effects of phenethyl isothiocyanate on N-nitrosobenzylmethylamine carcinogenesis in the rat esophagus.

F-344 rats fed diets containing phenethyl isothiocyanate (PEITC; 3 and 6 mumol/g diet), a naturally occurring constituent of cruciferous vegetables, before and during treatment with the carcinogen N-nitrosobenzylmethylamine (NBMA), developed 99-100% fewer esophageal tumors than NBMA-treated control rats. PEITC exhibited inhibitory effects against both preneoplastic lesions (acanthosis and hyperkeratosis, leukoplakia, leukokeratosis) and neoplastic lesions (papilloma, carcinoma). Tumors were not observed in rats treated with PEITC alone. The effects of PEITC (10, 25, 50, 100 microM) on the metabolism and DNA binding of NBMA in cultured explants of rat esophagus were also investigated. PEITC produced a marked (53-97%) dose-dependent inhibition in the binding of NBMA metabolites to DNA and in the levels of DNA methylation at the N7 (20-89%) and O6 (55-93%) positions of guanine. This isothiocyanate also reduced the metabolism of NBMA by esophageal tissues as indicated by increased amounts of unmetabolized NBMA in the medium of cultures containing PEITC. Collectively, these data indicate that PEITC is a potent inhibitor of NBMA-induced esophageal carcinogenesis in rats.

Deuterium isotope effect on the toxicokinetics of monomethylamine in the rat.

The single-dose toxicokinetics of monomethylamine has been characterized in the rat by HPLC assay of serial blood samples. Biphasic first-order elimination was observed following an iv bolus dose of 19 mumol/kg with a terminal half-life of 19.1 +/- 1.3 min (mean +/- SE, N = 4). The apparent steady state volume of distribution, systemic blood clearance, and renal blood clearance were 1.21 +/- 0.09 liter/kg, 53.4 +/- 3.5 ml/min/kg, and 5.72 +/- 0.53 ml/min/kg, respectively. The administration of an intragastric dose permitted the calculation of the systemic bioavailability of monomethylamine as 69 +/- 3%. Duplicate experiments using the structural analogue with deuterium atoms substituted for hydrogens on the methyl group revealed a much slower elimination of the compound, although ultimately, 5 times as much was excreted unchanged in the urine. Isotope effects calculated as the ratios of terminal half-life, systemic blood clearance, and systemic bioavailability were 1.9, 2.2, and 1.8, respectively.

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat.

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.

Single-dose toxicokinetics of N-nitrosomethylethylamine and N-nitrosomethyl (2,2,2-trideuterioethyl)amine in the rat.

To investigate the origins of an organotropic shift toward increasing esophageal carcinogenicity and DNA alkylation caused by beta-trideuteration of the hepatocarcinogen, N-nitrosomethylethylamine (NMEA), the single-dose toxicokinetics of NMEA and N-nitrosomethyl(2,2,2-trideuterioethyl)amine (NMEA-d3) has been characterized in 8-week-old male Fischer 344 rats by analysis using high performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to rats revealed biphasic first order elimination with a terminal half-life of 9.46 +/- 0.69 min for unchanged NMEA and 28.9 +/- 2.4 min for total radioactivity. Extensive conversion to polar metabolites was observed in the chromatograms. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMEA were 39.9 +/- 4.6 ml/min/kg and 496 +/- 36 ml/kg, respectively. There was negligible plasma protein binding and no detectable NMEA was excreted unchanged in the urine. Larger doses given by gavage indicated a systemic bioavailability of 25 +/- 1%. Similar doses of NMEA-d3 given to other groups of rats revealed no significant differences in any of the toxicokinetic parameters. No N-nitrosomethyl(2-hydroxyethyl)amine was found as a detectable metabolite of NMEA or NMEA-d3 in any of the blood or urine samples which were analyzed. When considered together, the data suggest that previously observed differences in organ specificity for the carcinogens, NMEA and NMEA-d3, are not due to differences in the total amounts of nitrosamine reaching particular tissues, but may have other localized causes such as differences in the enzymes responsible for metabolism which are present in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The Fenton degradation as a nonenzymatic model for microsomal denitrosation of N-nitrosodimethylamine.

The microsomal metabolism of the carcinogen N-nitrosodimethylamine (NDMA) was suggested to be initiated by hydrogen atom abstraction to form an alpha-nitrosamino radical, which either oxidizes further to an alpha-hydroxy nitrosamine as the initial product of the activating dealkylation pathway or fragments to the nitric oxide radical and N-methylformaldimine as the first step of the presumably inactivating denitrosation route. To examine the chemistry of the alpha-nitrosamino radical in a nonenzymatic setting, we exposed NDMA to the Fenton reagent, which is known to be capable of abstracting hydrogen atoms from organic species. The products observed were those expected of a denitrosation model. Solutions containing 13 mM [14C]NDMA, 15 mM FeSO4, 15 mM H2O2, and 7.5 mM H2SO4 were kept at 4-10 degrees C for 1 h and then basified to yield methylamine (3.2 +/- 0.5 mM, mean +/- SD, n = 8), formaldehyde (3.1 +/- 0.9 mM), and unreacted nitrosamine (10.2 +/- 0.7 mM) as the only radioactive species detected, with total nitrate/nitrite also being found at a level of 2.8 +/- 0.5 mM. N-Methylformaldiminium ion was identified as an intermediate. The parallels between these results and those seen in the microsomal reaction support the hypothesis that the alpha-nitrosamino radical is a common intermediate in enzymatic denitrosation versus dealkylation of NDMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in the single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine in the rat.

The single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine (NMHA) has been characterized in 8-week-old Fischer 344 rats by analysis using high-performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to male rats revealed biphasic first-order elimination with a terminal half-life of 37.4 +/- 1.7 min for unchanged NMHA and 101 +/- 6 min for total radioactivity, and extensive conversion to polar metabolites was seen in the high-performance liquid chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMHA were 13.1 +/- 0.9 ml/min/kg, and 685 +/- 31 ml/kg, respectively. Renal blood clearance and intrinsic hepatic clearance were estimated to be 0.805 +/- 0.024 and 16.7 +/- 2.1 ml/min/kg, respectively. A similar dose given to female rats yielded a terminal half-life for NMHA of 27.2 +/- 1.2 min, a steady-state volume of distribution of 652 +/- 23 ml/kg, and systemic blood, renal blood, and intrinsic hepatic clearances of 16.9 +/- 1.3, 1.45 +/- 0.14, and 22.5 +/- 0.3 ml/min/kg, respectively. The sex differences in terminal half-life and systemic blood, renal blood, and intrinsic hepatic clearances were significant at the P less than 0.05 level. Larger doses given by gavage, which appeared to be completely absorbed from the gut, indicated systemic bioavailabilities for unchanged NMHA of 78 +/- 10% and 69 +/- 1% for male and female rats, respectively. Binding of NMHA to plasma proteins was found to be negligible. Taken together the data allow for the conclusion that the observed sex differences in toxicokinetic parameters are due to differences in the intrinsic hepatic clearance of the compound. This difference in the ability of the liver to metabolize NMHA in vivo correlates with and may contribute to the greater susceptibility of female rats to hepatocarcinogenesis and of male rats to development of tumors in the nasal epithelium following oral exposure to NMHA.

Potential for metabolic deactivation of carcinogenic N-nitrosodimethylamine in vivo.

Enzymatic cleavage of N-nitrosodimethylamine (NDMA) to nitrite (normally representing about 10% of the total metabolism in vitro) also produces methylamine in yields roughly equimolar to those of nitrite, suggesting that the 'denitrosation' pathway may be responsible for the previously unexplained detection of methylamine as a urinary metabolite of NDMA and, at least in part, for the recovery of less than stoichiometric amounts of dinitrogen in 15N-labelling experiments. We have now followed excretion of labelled methylamine by rats receiving 14C-NDMA as a possible index of the extent of in-vivo denitrosation. Correcting for the proportion of labelled methylamine recovered in the urine following its administration under the conditions used for NDMA, 2.5-10% of the NDMA metabolism in Fischer rats appeared to proceed by a methylamine-forming route. The results are consistent with the conclusion that the metabolism of NDMA is best viewed as a competition between two pathways, with denitrosation diverting a significant proportion of the clearance to a presumably deactivating metabolic route at the expense of the activating alkylation pathway responsible for carcinogenesis.

[Immunostimulant action of polysaccharides (heteroglycans) from higher plants. Preliminary communication]. / Immunstimulierend wirkende Polysaccharide (Heteroglykane) aus höheren Pflanzen. Vorläufige Mitteilung.

From the water or alcaline-water extracts of Echinacea purpurea (L.) Moench and -angustifolia DC., Eupatorium cannabinum L. and -perfoliatum L., Chamomilla recutita (L.) (Rauscher), Calendula officinalis L., Baptisia tinctoria (L.) R.B., Achyrocline satureoides DC., Arnica montana L., Sabal serrulata Roem et Schult. and Eleutherococcus senticosus Maxim. polysaccharide fractions with molecular weights in the range of 25 000 to 500 000 and higher have been isolated, which, according to the granulocytes- and carbon clearance tests, showed significant immunostimulating activities. The isolated compounds belong to the group of water-soluble, acidic heteroglycanes. The linkages in the different polysaccharides do not represent a uniform structure type.

[DC- and HPLC-Analysis of Eleutherococcus.]. / Die DC- and HPLC-Analyse der Eleutherococcus Droge.

A rapid and reproducible TLC- and HPLC-analysis of root-extracts and preparations of ELEUTHEROCOCCUS SENTICOSUS on the basis of the characteristic lignans and other phenylpropan derivatives has been developed.