Increased synthesis of a new oleanane-type saponin in hairy roots of marigold (Calendula officinalis) after treatment with jasmonic acid.

Native plant of marigold (Calendula officinalis L.) synthesizes oleanolic acid saponins classified as glucosides or glucuronides according to the first residue in sugar chain bound to C-3 hydroxyl group. Hairy root culture, obtained by transformation with Agrobacterium rhizogenes strain 15834, exhibit a potent ability of synthesis of oleanolic acid glycosides. The HPLC profile of saponin fraction obtained from C. officinalis hairy roots treated with plant stress hormone, jasmonic acid, showed the 10-times increase of the content of one particular compound, determined by NMR and MALDI TOF as a new bisdesmoside saponin, 3-O-ß-d-glucuronopyranosyl-28-O-ß-d-galactopyranosyl-oleanolic acid. Such a diglycoside does not occur in native C. officinalis plant. It is a glucuronide, whereas in the native plant glucuronides are mainly accumulated in flowers, while glucosides are the most abundant saponins in roots. Thus, our results revealed that the pathways of saponin biosynthesis, particularly reactions of glycosylation, are altered in C. officinalis hairy root culture.

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes.

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent (12.2 +/- 0.2 and 7.5 +/- 0.2%, respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.

Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate.

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are the major polyphenolic constituents in green tea. In this study, we characterized the enzymology of cytosolic catechol-O-methyltransferase (COMT)-catalyzed methylation of EGCG and EGC in humans, mice, and rats. At 1 microM, EGCG was readily methylated by liver cytosolic COMT to 4"-O-methyl-EGCG and then to 4',4"-di-O-methyl-EGCG; EGC was methylated to 4'-O-methyl-EGC. The K(m) and V(max) values for EGC methylation were higher than EGCG; for example, with human liver cytosol, the K(m) were 4.0 versus 0.16 microM and V(max) were 1.28 versus 0.16 nmol/mg/min. Rat liver cytosol had higher COMT activity than that of humans or mice. The small intestine had lower specific activity than the liver in the methylation of EGCG and EGC. Glucuronidation on the B-ring or the D-ring of EGCG greatly inhibited the methylation on the same ring, but glucuronidation on the A-ring of EGCG or EGC did not affect their methylation. Using EGC and 3,4-dihydroxy-L-phenylalanine as substrates, EGCG, 4"-O-methyl-EGCG, and 4',4"-di-O-methyl-EGCG were all potent inhibitors (IC(50) approximately 0.2 microM) of COMT. The COMT-inhibiting activity of (-)-EGCG-3'-O-glucuronide was similar to EGCG, but (-)-EGCG-4"-O-glucuronide was less potent. The present work provides basic information on the methylation of EGCG and suggests that EGCG may inhibit COMT-catalyzed methylation of endogenous and exogenous compounds.

Development of an in vitro screening model for the biosynthesis of acyl glucuronide metabolites and the assessment of their reactivity toward human serum albumin.

An in vitro screening model was developed to determine the reactivity of acyl glucuronide metabolites from carboxylic drugs. This assay is composed of two phases. The first is a phase of biosynthesis of acyl glucuronides by human liver microsomes (HLM). The second, during which acyl glucuronides are incubated with human serum albumin (HSA), consists of assessing the reactivity of acyl glucuronides toward HSA. Both phases are performed successively in the same experiment. This model was validated using eight carboxylic drugs that were well known for their reactivity, their extent of covalent binding, and their immunological potential. These products were representative of the scale of reactivity. Each compound was incubated with HLM at 400 microM and metabolized into acyl glucuronide to different extents, ranging from 5.6% (tolmetin) to 89.4% (diclofenac). The first-order aglycone appearance rate constant and the extent of covalent binding to proteins were assayed during the incubation of acyl glucuronides formed with HSA for 24 h. Extensive isomerization phenomenon was observed for each acyl glucuronide between the two phases. An excellent correlation was observed (r(2), 0.94) between the extent of drug covalent binding to albumin and the aglycone appearance constant weighted by the percentage of isomerization. This correlation represents an in vitro reactivity scale, which will be helpful in drug discovery support programs to predict the covalent binding potential of new chemical entities. This screening model will also allow the comparison of acyl glucuronide reactivity for related structure compounds.

Green tea and the metabolism of 2-amino-3-methylimidazo.

The effects of green tea intake on the metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the rat was studied. IQ belongs to a new class of mutagens and carcinogens, heterocyclic arylamines, formed during cooking through browning meats and fish, thus, in the food chain of most non-vegetarians. Ten adult male and female Fischer 344 rats were placed on a 2% solution of green tea and 10 control rats were on water for 6 weeks. Then, animals were administered a single dose of 40 mg/kg body weight of [2-14C]IQ by oral gavage. Twenty-four hour urine samples were collected and metabolites were separated by HPLC and quantitated by scintillation counting. Two minor and three major metabolites were isolated, including, small quantities of IQ itself. The rats on tea showed significant differences (P < 0.05) in the recovery of the three major metabolites, namely, IQ-sulfamate, IQ-5-O-sulfate, and IQ-5-O-glucuronide, respectively. Green tea, therefore, influences the manner in which the food carcinogen IQ is metabolized and excreted in urine. Formation of glucuronides, increased by green tea, represent a key means of detoxification of the heterocyclic amine, IQ.